Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational I
Grant Number: 
TR1-01267
Investigator: 
Type: 
Partner-PI
ICOC Funds Committed: 
$5 416 003
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Victoria, Australia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Parkinson's Disease (PD) is a devastating disorder, stealing vitality from vibrant, productive adults & draining our health care dollars. It is also an excellent model for studying other neurodegenerative conditions. We have discovered that human neural stem cells (hNSCs) may exert a significant beneficial impact in the most authentic, representative, & predictive animal model of actual human PD. Interestingly, we have learned that, while some of the hNSCs differentiate into replacement dopamine (DA) neurons, much of the therapeutic benefit derived from a stem cell action we discovered a called the “Chaperone Effect” – even hNSC-derived cells that do not become DA neurons contributed to the reversal of severe Parkinsonian symptoms by protecting endangered host DA neurons & their connections, restoring equipoise to the host nigrostriatal system, and reducing pathological hallmark of PD. While the ultimate goal may someday be to replace dead DA neurons, the Chaperone Effect represents a more tractable near-term method of using cells to address this serious condition. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest, most efficacious) way to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From skin cells reprogrammed to act like stem cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of stem cell-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This study seeks to answer these questions. Importantly, we will do so using the most representative model of human PD, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. A successful therapy for PD would not only be of great benefit for the many patients who now suffer from the disease, or who are likely to develop it as they age, but the results will help with other potential disease applications due to greater understanding of stem cell biology (particularly the Chaperone Effect, which represents “low hanging fruit”) as well as their potential complications and side effects.
Statement of Benefit to California: 
Not only is Parkinson's Disease (PD) a devastating disease in its own right-- impairing typically vibrant productive adults & draining our health care dollars -- but it is also an excellent model for studying other neurodegenerative diseases. We have discovered that stem cells may actually exert a beneficial impact independent of dopamine neuron replacement. As a result of a multiyear study performed by our team, implanting human neural stem cells (hNSCs) into the most authentic, representative, and predictive animal model of actual human PD, we learned that the cells could reverse severe Parkinsonian symptoms by protecting endangered host dopaminergic (DA) neurons, restoring equipoise to the cytoarchitecture, preserving the host nigrostriatal pathway, and reducing alpha-synuclein aggregations (a pathological hallmark of PD). This action, called the "Chaperone Effect" represents a more tractible near-term method of using cells to address an unmet medical need. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest & most efficacious way) to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From human induced pluripotent cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of donor-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This study seeks to answer these questions. Importantly, we will continue to use the most representative model of human PD to do so, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. Because of the unique team enlisted, these studies can be done at a fraction of the normal cost, allowing for parsimony in the use of research dollars, clearly a benefit to California taxpayers. Not only might California patients benefit in terms of their well-being, and the economy benefit from productive adults re-entering the work force & aging adults remaining in the work force, but it is likely that new intellectual property will emerge that will provide additional financial benefit to California stakeholders, both citizens & companies.
Progress Report: 
  • Parkinson's Disease (PD) is a devastating disorder, stealing vitality from vibrant, productive adults & draining our health care dollars. It is also an excellent model for studying other neurodegenerative conditions. We have discovered that human neural stem cells (hNSCs) may exert a significant beneficial impact in the most authentic, representative, & predictive animal model of actual human PD (the adult African/St. Kitts Green Monkeys exposed systemically to the neurotoxin MPTP). Interestingly, we have learned that, while some of the hNSCs differentiate into replacement dopamine (DA) neurons, much of the therapeutic benefit derived from a stem cell action we discovered called the “Chaperone Effect” – even hNSC-derived cells that do not become DA neurons contributed to the reversal of severe Parkinsonian symptoms by protecting endangered host DA neurons & their connections, restoring equipoise to the host nigrostriatal system, and reducing pathological hallmark of PD. While the ultimate goal may someday be to replace dead DA neurons, the Chaperone Effect represents a more tractable near-term method of using cells to address this serious condition. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest, most efficacious) way to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From skin cells reprogrammed to act like stem cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of stem cell-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This international study – which involves scientists from California, Madrid, Melbourne -- has been seeking to answer these questions. Importantly, we have been doing so using the most representative model of human PD, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. A successful therapy for PD would not only be of great benefit for the many patients who now suffer from the disease, or who are likely to develop it as they age, but the results will help with other potential disease applications due to greater understanding of stem cell biology (particularly the Chaperone Effect, which represents “low hanging fruit”) as well as their potential complications and side effects.
  • To date, we have transplanted nearly 40 Parkinsonian non-human primates (NHPs) with a range of the different stem cell types described above. We have been able to generate neurons from some of these stem cells that appear to have the characteristics of the desired A9-type midbrain dopaminergic neuron lost in PD. Following transplantation, some of these stem cell derivatives appear to survive, integrate, & behave like dopaminergic neurons. Preliminary behavioral analysis of some engrafted NHPs offers encouraging results, suggesting an improvement in the Parkinsonism score in some of the animals. These NHPs will need to be followed for 1 year to insure that improvement continues & that no adverse events intervene. Over the next year, more stem cell candidates will be tested as we further optimize their preparation & differentiation.
  • We have made substantial progress in what will amount to the largest and most comprehensive head-to-head behavioral analysis of stem cell transplanted MPTP-NHPs to date and have identified cell types that show dramatic improvement in this model. Compared to the improvement observed with undifferentiated fetal CNS-derived hNSCs (the stem cell type in used Redmond et al, PNAS, 2007), 3 human stem cell candidates have shown a larger improvement in PS.
  • Summary of Achievements for this reporting period
  • • Comprehensive Behavioral data collection of 84 monkeys comprising over 10,000 observation data points
  • • Statistical analysis of Behavioral data collected to date identifies striking and statistically significant improvements in PS for several stem cell types. (Accordingly, NO-GO (or near NO-GO) cell types have been identified via comparison of levels of improvement or no improvement) [Figure 1]
  • • DNA samples collected in order to pursue the first ever complete genome sequencing of the Vervet in collaboration with the Washington University Genome Center
  • • Biochemistry sample processing and data collection of a 2nd large batch of samples completed.
  • The identification and development of an ideal cell-based therapy for a complex neurodegenerative disease requires the rigorous evaluation of both efficacy and safety of different sources and subtypes of hNSCs. The objective of this project has been to fully evaluate and identify the optimal stem cell type for a cell based therapy for refractory Parkinson’s Disease (PD) using the systemically MPTP-lesioned Old World non-human primate (NHP) (the St. Kitts Green Monkey) the most authentic animal model of the actual human disease. Among a list of plausible potentially therapeutic stem cell sources, 7 candidates have been evaluated head-to-head. The intent has been that the stem cell type (and its derivatives) safely producing the largest improvement in behavioral scores (based on a well-established NHP PD score – the Parkinson’s Factor Score [PFS] or ParkScore (which closely parallels the Hoehn–Yahr scale used in human patients, and is an accurate functional read-out of nigrostriatal dopamine [DA] activity) -- as well as a Healthy Behaviors Score [HBS] (similar to the activities-of-daily-living [ADL] on the major Parkinson’s rating scale and allows quantification of adverse events) -- will be advanced towards IND-enabling studies, to an actual IND filing, and ultimately a clinical trial.
  • Candidate cells have been transplanted into specific sub-regions of the nigrostriatal pathway of MPTP-lesioned NHPs. Animals undergo behavioral scoring for analysis of severity of Parkinsonian behavior at multiple time points pre- and post-cell transplantation. At sacrifice, biochemical measurements of DA content are made. Tissue is also analyzed to determine the fate of donor cells; the status of the host nigrostriatal pathway; the number of alpha-synuclein aggregates; degree of inflammation; any evidence of adverse events (e.g., tumor formation, cell overgrowth, emergence of cells inappropriate to the CNS).
  • We have made substantial progress in what will amount to the largest and most comprehensive head-to-head analysis of stem cell transplanted into any disease model to date, let alone behavioral analysis into a primate model of PD. Behavioral data have been collected on ~100 monkeys comprising >10,000 observation data points. We have identified a single Developmental Candidate (DC) that shows consistent and dramatic improvement in severely Parkinsonian NHPs (i.e., a significant decrease in Parkinsonian symptoms over the entire evaluation period), reflecting a restitution of DA function – human embryonic stem cell (hESC-derived) ventral mesencephalic (VM) precursors. We also suggest adding a mechanism to these cells for insuring unambiguous safety and invariant lineage commitment (a construct already generated and inserted into this DC, and recently engrafted into some initial monkeys).
  • We believe are ready for IND-enabling studies, including additional long-term pre-clinical behavioral studies of hESC-derived hVM cells that bear the above-mentioned “safety construct” – combined with additional biochemical assays of DA metabolism, histological assessments, serial profiling to insure genomic stability. Scale-up conditions for this DC are defined and reproducible and a working cell bank has been established.
  • Parkinson's Disease (PD) is a devastating disorder that is caused by the loss of a particular type of neuron in the brain. PD patients show movement abnormalities which worsen over time and significantly reduce the quality of life. Current treatments reduce the severity of these problems but very often the efficacy of these treatments gradually weakens over time leaving patients with few therapeutic options, some of which carry significant unwanted side effects. Since the development of growing undifferentiated human stem cells in the late 1990’s, much has been learned in regards to how to make these cells develop into neuronal cells, in particular the same type of neuron that is lost in a PD patient. Therefore, a cellular therapy has been envisioned for the treatment of PD, however, the complex nature of this disease requires higher level models in which potential therapies can be accurately evaluated before moving a therapy to clinical trials.
  • Previous work using human fetal tissue showed improvement of PD symptoms in an animal model and human clinical trials, however, distinctive movement abnormalities arose from the use of this treatment and combined with the ethical issues, it is not a viable therapeutic strategy. Recent work suggests that the use of embryonic stem cells for the treatment of PD may be possible but a direct comparison of the different types of cells derived from these was lacking. Additionally, tumors caused by these cells have been reported.
  • Our research efforts funded by this CIRM award allowed us to complete the largest stem cell therapy comparison for PD using the most accurate disease model available. Over the last 3 years we have evaluated the efficacy of 8 potential therapeutic cell types and 2 control cell types (in addition to various other control groups to rule out any possibility that the observations may have resulted from something other than cells). From these efforts we have confidently identified a strategy for producing cells that show a dramatic reduction in the PD symptoms in this model and these cells will be developed for clinical trials. Furthermore, we have incorporated a critical step for ensuring the safety of this cell therapy by including a purification technique that removes cells that may give rise to tumors or produce unknown or unwanted effects.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01257
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$2 753 559
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
One in every ten thousand people in the USA have Huntington's Disease, and it impacts many more. Multiple generations within a family can inherit the disease, resulting in escalating health care costs and draining family resources. This highly devastating and fatal disease touches all races and socioeconomic levels, and there are currently no cures. Screening for the mutant HD gene is available, but the at-risk children of an affected parent often do not wish to be tested since there are currently no early prevention strategies or effective treatments. HD is a challenging disease to treat. Not only do the affected, dying neurons need to be salvaged or replaced, but also the levels of the toxic mutant protein must be diminished to prevent further neural damage and to halt progression of the movement disorders and physical and mental decline that is associated with HD. Our application is focused on developing a safe and effective therapeutic strategy to reduce levels of the harmful mutant protein in damaged or at-risk neurons. We are using an RNA interference strategy – “small interfering RNA (siRNA)” to prevent the mutant protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However, the inability to deliver the therapeutic molecules into the human brain in a robust and durable manner has thwarted scale-up of this potentially curative therapy into human trials. We are using mesenchymal stem cells, the “paramedics of the body”, to deliver the therapeutic siRNA directly into damaged cells. We have discovered that these stem cells are remarkably effective delivery vehicles, moving robustly through the tissue and infusing therapeutic molecules into each damaged cell that they contact. Thus we are utilizing nature's own paramedic system, but we are arming them with a new tool to also reduce mutant protein levels. Our novel system will allow the therapy to be carefully tested in preparation for future human cellular therapy trials for HD. The significance of our studies is very high because there are currently no treatments to diminish the amount of toxic mutant htt protein in the neurons of patients affected by Huntington’s Disease. There are no cures or successful clinical trials for HD. Our therapeutic strategy is initially examining models to treat HD, since the need is so acute. But this biological delivery system could also be used, in the future, for other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA1), Alzheimer's Disease, and some forms of Parkinson's Disease, where reduction of the levels of a mutant or disease-activating protein could be curative. Development of this novel stem cell therapeutic and effective siRNA delivery system is extremely important for the community of HD and neurodegenerative disease researchers, patients, and families.
Statement of Benefit to California: 
It is estimated that one in 10,000 CA residents have Huntington’s Disease (HD). While the financial burden of Huntington’s Disease is estimated to be in the billions, the emotional burden on the friends and families of HD patients is immeasurable. Health care costs are extremely high for HD patients due to the decline in both body and mind. The lost ability of HD patients to remain in the CA workforce and to support their families causes additional financial strain on the state’s economy. HD is inherited as an autosomal dominant trait, which means that 50% of the children of an HD patient will inherit the disease and will in turn pass it on to 50% of their children. Individuals diagnosed through genetic testing are at risk of losing insurance coverage. Since there are currently no cures or successful clinical trials for HD, many are reluctant to be tested. The proposed project is designed in an effort to reach out to these individuals who, given that HD is given an orphan disease designation, may feel that they are completely forgotten and thus have little or no hope for their future or that of their families. To combat this devastating disease, we are using an RNA interference strategy, “small interfering RNA (siRNA),” to prevent the mutant htt protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However the siRNA needs to be delivered to the brain or central nervous system in a continual manner, to destroy the toxic gene products as they are produced. There are currently no methods to infuse or produce siRNA in the brain, in a safe and sustained manner. Therefore the practical clinical use of this dramatically effective potential therapeutic application is currently thwarted. Here we propose a solution, using adult mesenchymal stem cells (MSC) modified to infuse siRNA directly into diseased or at-risk neurons in the striata of HD patients, to decrease the levels of the toxic mutant htt protein. MSC are known as the “paramedics of the body" and have been demonstrated through clinical trials to be safe and to have curative effects on damaged tissue. Even without the modification to reduce the mutant protein levels, the infused MSC will help repair the damaged brain tissue by promoting endogenous neuronal growth through secreted growth factors, secreting anti-apoptotic factors, and regulating inflammation. Our therapeutic strategy will initially examine models to treat HD, since the need is so acute. But our biological delivery system could also be applied to other neurodegenerative disorders such as ALS, some forms of Parkinson’s Disease, and Alzheimer’s Disease, by using siRNA to interfere with key pathways in development of the pathology. This would be the first cellular therapy for HD patients and would have a major impact on those affected in California. In addition, the methods that we are developing will have far-reaching effects for other neurodegenerative disorders.
Progress Report: 
  • During the first year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • This year we have shown that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs through direct cell-to-cell transfer of siRNA, and we have filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. A manuscript documenting the results of these studies is in preparation.
  • We continue to explore the precise methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-protein interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project and a collaborative manuscript is in preparation.
  • During year two of the proposed studies we will continue to document levels of reduction of the toxic htt protein in different types of neurons, including medium spiny neurons (MSN) derived from HD patient induced pluripotent stem cells (iPSC). We have made significant advances in developing the tools for these studies, including HD iPSC line generation and MSN maturation from human pluripotent cells in culture. A manuscript on improved techniques for generating MSN from pluripotent cells is in preparation. We have also worked closely with our colleagues at the UC Davis MIND Institute to achieve improved maturation and electrical activity in neurons derived from human pluripotent stem cells in vitro, and we are examining the impact of human MSC on enhancing survival of damaged human neurons.
  • In the second year of funding we will test efficacy of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of our newly developed strain of immune deficient Huntington's disease mice. This strain was developed by our teams at UC Davis to allow testing of human cells in the mice, since the current strains of HD mice will reject human stem cells. A manuscript describing generation of this novel HD mouse strain is in preparation, in collaboration with our nationally prominent Center for Mouse Biology.
  • Behavioral studies will be conducted in this strain with and without the MSC/siRNA-mediated knockdown of the mutant protein, through years 2-3, in collaboration with our well established mouse neurobehavioral core at the UC Davis Center for Neurosciences. We have documented the safety of intrastriatal injection of human MSC in immune deficient mice and will next test the efficacy of human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo.
  • As added leverage for this grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • From the funded studies 4 manuscripts are now in preparation, a chapter is in press and a review paper on MSC to treat neurodegenerative diseases is in press.
  • During the second year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. During the second year we have more fully characterized our development candidate; MSC/anti-htt. We have documented that normal human donor MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. We reported this work at the Annual meeting of the American Academy of Neurology (G Mitchell, S Olson, K Pollock, A Kambal, W Cary, K Pepper, S Kalomoiris, and J Nolta. Mesenchymal Stem Cells as a Delivery Vehicle for Intercellular Delivery of RNAi to Treat Huntington's disease. AAN IN10-1.010, 2011) and have recently completed and submitted a manuscript describing these results (S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Submitted, In Review, July 2011).
  • We have explored the molecular methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-siRNA interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project. This work was also presented at AAN 2011, and a collaborative manuscript is in preparation for submission (S Olson, G McNerny, K Pollock, F Chuang, T Huser and J Nolta, Visualization of siRNA Complexed to RISC Machinery: Demonstrating Intercellular siRNA Transfer by Imaging Activity. MS in preparation, Presented at AAN 2011: IN4-1.014).
  • In the second year of funding we developed the models for in vivo efficacy testing of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of established and new strains of Huntington's disease mice. Behavioral studies were conducted in two strains, the R6/2 immune competent mice and our new immune deficient strain, the NSG/HD, in comparison to normal littermate controls that are not affected by HD. We established the batteries of behavioral tests that are now needed to test efficacy of our development candidate in the brain, in year three. Established tests include rotarod, treadscan, pawgrip, spontaneous activity, nesting, locomotor activity, and the characteristic HD mouse hindlimb clasping phenotype. In addition we monitor the status of weight and tremor, grooming, eyes, hair, body position, and tail position, which all change over time in HD mice. These tests are conducted at 48 hour intervals by two highly trained technicians who are blinded to the treatment that the mouse had received. These behavioral and phenotypic tests have been established at the level of Good laboratory practices in our new Institute for Regenerative Cures shower-in barrier facility vivarium. We have documented the biosafety of intrastriatal injection of human MSC in immune deficient mice and are now examining the in vivo efficacy of the development candidate: human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo, which will be completed in year three.
  • As added leverage for this funded grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • During the three years of funding we made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • We initially demonstrated that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neuronal cells in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs either through direct cell-to-cell transfer of siRNA or through exosome transfer, and we filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. This patent has IP sharing with CIRM.
  • An NIH transformative grant was awarded to Dr. Nolta to further explore these exciting findings. This provides funding for five years to further define and optimize the siRNA transfer mechanism.
  • A manuscript documenting the results of these studies was published:
  • S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience; 49(3):271-81, 2012.
  • Also a review was published with our collaborator Dr. Gary Dunbar:
  • S Olson, K Pollock, A Kambal, W Cary, G Mitchell, J Tempkin, H Stewart, J McGee, G Bauer, T Tempkin, V Wheelock, G Annett, G Dunbar and J Nolta, Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s disease. Molecular Neurobiology; 45(1):87-98, 2012.
  • We examined the potential efficacy of injecting relatively small numbers of MSCs engineered to produce ant-htt siRNA into the striata of the HD mouse strain R6/2, in three series of experiments. Results of these experiments did not reach significance for the test agent as compared to controls. The slope of the decline in rotarod performance was less with the test agent, and development of clasping behavior was slightly delayed after injection of MSC/aHtt, but this caught up to the controls and was not significant after day 60.
  • Our conclusions are that the R6/2 strain is too rapidly progressing to see efficacy with the test agent, and also that improved methods of siRNA transfer from cell to cell are needed. We are currently working on this problem through the NIH transformative award, and will use the YAC 128 strain, which has a more slowly progressing phenotype, for all future studies. These mice are now bred and in use in our vivarium, for the MSC/BDNF studies funded through our disease team grant.
  • Through this translational grant funding we have also developed in vitro potency assays, using human embryonic stem cell-derived neurons and medium spiny neurons, as we have described in prior reports. The differentiation techniques (funded through other grants to our group) have now been published:1-3
  • 1. Liu J, Githinji J, McLaughlin B, Wilczek K, Nolta J. Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell-Derived Neural Stem Cells. Stem Cell Rev;8(4):1129-37, 2012.
  • 2. Jun-feng Feng, Jing Liu, Xiu-zhen Zhang, Lei Zhang, Ji-yao Jiang, Nolta J, Min Zhao. Guided Migration of Neural Stem Cells Derived from Human Embryonic Stem Cells by an Electric Field. Stem Cells. Feb; 30(2):349-55, 2012.
  • 3. Liu J, Koscielska KA, Cao Z, Hulsizer S, Grace N, Mitchell G, Nacey C, Githinji J, McGee J, Garcia-Arocena D, Hagerman RJ, Nolta J, Pessah I, Hagerman PJ. Signaling defects in iPSC-derived fragile X premutation neurons. Hum Mol Genet. 21(17):3795-805. 2012.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01246
Investigator: 
Type: 
PI
Type: 
Partner-PI
ICOC Funds Committed: 
$3 701 766
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The goals of this study are to develop patient-specific induced pluripotent cell lines (iPSCs) from patients with Parkinson’s disease (PD) with defined mutations and sporadic forms of the disease. Recent groundbreaking discoveries allow us now to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These lines will be used as an experimental pre-clinical model to study disease mechanisms unique to PD. We predict that these cells will not only serve an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are pathologically affected with PD. The specific objectives of these studies are to (1) establish a bank of iPSCs from patients with idiopathic PD and patients with defined mutations in genes associated with PD, (2) differentiate iPSCs into dopaminergic neurons and assess neurochemical and neuropathological characteristics of PD of these cells in vitro, and (3) test the hypothesis that specific pharmacologic agents can be used to block or reverse pathological phenotypes. The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of PD, which, if solved in this collaborative effort, would be instantly translated into a wide range of clinical applications, including drug discovery. This research is highly translational, as the final component is aimed at testing lead compounds that could be neuroprotective, and ultimately at developing a high-throughput drug screening program to discover new disease modifying compounds. 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: 
Approx. 36,000-60,000 people in the State of California are affected with Parkinson’s disease (PD), a common neurodegenerative disease that causes a high degree of disability and financial burden for our health care system. It is estimated that the number of PD cases will double by the year 2030. We have a critical need for novel therapies that will prevent or even reverse neuronal cell loss of specific neurons in the brain of patients. This collaborative proposal will provide real benefits and values to the state of California and its citizens in providing new approaches for understanding disease mechanisms, diagnostic tools and drug discovery of novel treatment for PD. Reprogramming of adult skin cells to a pluripotent state is the underlying mechanism upon which this application is built upon and offers an attractive avenue of research in this case to develop an ‘authentic’ pre-clinical model of PD. The rationale for the proposed research is that differentiated pluripotent stem cells from patients with known genetic forms of PD will recapitulate in vitro one or more of the key molecular aspects of neural degeneration associated with PD and thus provide an entirely novel human cellular system for investigation PD-related disease pathways and for drug discovery. The impact of this collaborative research project, if successful, is difficult to over-estimate. The scientific field has been struggling with the inability to directly access cells that are affected by the disease process that underlies PD and therefore all research and drug discovery has relied on ”best guess” models of the disease. Thus, the absence of cellular models of Parkinson’s disease represents a huge bottleneck in the field.
Progress Report: 
  • In the first year of the CIRM Early translational research award, we established a bank of 51 cell lines derived from skin cells of patients with Parkinson’s disease that carry specific mutations in known genes that cause PD as well as sporadic PD patients. We also recruited matched healthy individuals that serve as controls.
  • In a next step, we reprogrammed (‘rejunivated’) 17 samples of skin cells to derive pluripotent stem cells (iPSC) that closely resemble human embryonic stem cells characterized by biochemical and molecular techniques. We also optimize this process by introducing factors the will be removed after successful reprogramming.
  • We have now built a foundation for the next milestones and made already progress on the differentiation into authentic dopamine producing cells, and we have developed assays to assess the Parkinson’s disease-specific pathological phenotype of the dopamine neurons.
  • The goal of this CIRM early translational grant is to develop a model for “Parkinson’s disease (PD) in a culture dish” using patient-specific induced pluripotent stem cell lines (iPS). The underlying idea is to utilize these lines as an experimental pre-clinical model to study disease mechanisms unique to PD that could lay the foundation for drug discovery.
  • Over the last year, we have expanded our patient skin cell bank to 57 cell lines and the iPS cell bank to 39 well-characterized pluripotent stem cell lines from PD patients and healthy controls individuals. We have improved current protocols of neuronal differentiation from patient-derived iPS lines into dopamine producing neurons and can show consistency and reproducibility of making midbrain dopamine expressing nerve cells.
  • In our first publication (Nguyen et al. 2011), we describe for the first time differences in iPS-derived neurons from a PD patient with a common causative mutation in the LRRK2 gene. These patient cells are more susceptible for cellular toxins leading ultimately to more cell degeneration and cell death.
  • We are also investigating a common disease mechanism implicated in PD, which is mitochondrial dysfunction. In skin cells of a patient we were able to find profound deficits of mitochondrial function compared to control lines and we are now in the process of confirming these results in neural precursors and mature dopamine neurons.
  • Overall, we have made substantial progress towards the goal of this grant which is the a new cell culture model of PD which can replicate PD-related cellular pathology.
  • The goal of this CIRM early translational grant is to develop a model for “Parkinson’s disease (PD) in a culture dish” using patient-specific induced pluripotent stem cell lines (iPS). The underlying idea is to utilize these lines as an experimental pre-clinical model to study disease mechanisms unique to PD that could lay the foundation for drug discovery.
  • Over the last year, we have expanded our patient skin cell bank to 61 cell lines and the iPS cell bank to 51 well-characterized pluripotent stem cell lines from PD patients and healthy controls individuals. We have improved current protocols of neuronal differentiation from patient-derived iPS lines into dopamine producing neurons and can show consistency and reproducibility of making midbrain dopamine expressing nerve cells. This has been now published in Mak et al. 2012. Furthermore, we also develop new protocols to also derive other neuronal subtypes and glia, which are the support cells in the brain, to build co-culture systems. These co-cultures might represent closer the physiological conditions in the brain.
  • In our first publication (Nguyen et al. 2011), we describe for the first time differences in iPS-derived neurons from a PD patient with a common causative mutation in the LRRK2 gene. These patient cells are more susceptible for cellular toxins leading ultimately to more cell degeneration and cell death. In a second publication Byers et al. 2011, we describe similar findings for a different mutation in the alpha-synuclein gene where the normal protein is overexpressed due to a triplication of the gene locus.
  • We are also investigating a common disease mechanism implicated in PD, which is mitochondrial dysfunction. In skin cells of a patient we were able to find profound deficits of mitochondrial function compared to control lines and we are now in the process of confirming these results in neural precursors and mature dopamine neurons.
  • We are expanding the assay development to other disease-related mechanisms such as deficits in outgrowth of neuronal projections and protein aggregation.
  • Overall, through this program we have developed an invaluable resource of patient-derived cell lines that will be crucial for understanding disease mechanisms and drug discovery. We also showed proof that these cell lines can indeed recapitulates important aspects of disease and are therefore valuable assets as research tools.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01245
Investigator: 
Type: 
PI
Institution: 
Type: 
Partner-PI
ICOC Funds Committed: 
$3 599 997
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Collaborative Funder: 
Victoria, Australia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer disease (AD), the most common cause of dementia among the elderly and the third leading cause of death, presently afflicts over 5 million people in the USA, including over 500,000 in California. Age is the major risk factor, with 5% of the population over age 65 affected, with the incidence doubling every 5 years thereafter, such that 40-50% of those over age 85 are afflicted. Being told that one suffers from AD is one of the most devastating diagnoses a patient (and their family/caregivers) can ever receive, dooming the patient to a decade or more of progressive cognitive decline and eventual loss of all memory. At the terminal stages, the patients have lost all reasoning ability and are usually bed-ridden and unable to care for themselves. As the elderly represent the fastest growing segment of our society, there is an urgent need to develop therapies to delay, prevent or treat AD. If the present trend continues and no therapy is developed, over 16 million Americans will suffer from AD by 2050, placing staggering demands on our healthcare and economic systems. Thus, supporting AD research is a wise and prudent investment, particularly focusing on the power that stem cell biology offers. Currently, there is no cure or means of preventing AD. Existing treatments provide minor symptomatic relief– often associated with severe side effects. Multiple strategies are likely needed to prevent or treat AD, including the utilization of cell based approaches. In fact, our preliminary studies indicate that focusing on the promise of human stem cell biology could provide a meaningful therapy for a disease for which more traditional pharmaceutical approaches have failed. We aim to test the hypothesis that neural stem cells represent a novel therapeutic strategy for the treatment of AD. Our broad goal is to determine whether neural stem cells can be translated from the bench to the clinic as a therapy for AD. This proposal builds on extensive preliminary data that support the feasibility of neural stem cell-based therapies for the treatment of AD. Thus, this proposal focuses on a development candidate for treating Alzheimer disease. To translate our initial stem cell findings into a future clinical application for treating AD, we assembled a world class multi-disciplinary team of scientific leaders from the fields of stem cell biology, animal modeling, neurodegeneration, immunology, genomics, and AD clinical trials to collaborate in this early translational study aimed at developing a novel treatment for AD. Our broad goal is to examine the efficacy of human neural stem cells to rescue the cognitive phenotype in animal models of AD. Our studies aim to identify a clear developmental candidate and generate sufficient data to warrant Investigational New Drug (IND) enabling activity. The proposed studies represent a novel and promising strategy for treating AD, a major human disorder for which there is currently no effective therapy.
Statement of Benefit to California: 
Neurological disorders have devastating consequences for the quality of life, and among these, perhaps none is as dire as Alzheimer disease. Alzheimer disease robs individuals of their memory and cognitive abilities, such that they are no longer able to function in society or even interact with their family. Alzheimer disease is the most common cause of dementia among the elderly and the most significant and costly neurological disorder. Currently, 5.2 million individuals are afflicted with this insidious disorder, including over 588,000 in the State of California. Hence, over 10% of the nation's Alzheimer patients reside in California. Moreover, California has the dubious distinction of ranking first in terms of states with the largest number of deaths due to this disorder. Age is the major risk factor for Alzheimer disease, with 5% of the population over age 65 afflicted, with the incidence doubling every 5 years such that 40-50% of the population over age 85 is afflicted. As the elderly represent the fastest growing segment of our society, there is an urgent need to develop therapies to prevent or treat Alzheimer disease. By 2030, the number of Alzheimer patients living in California will double to over 1.1 million. All ethnic groups will be affected, although the number of Latinos and Asians living with Alzheimer will triple by 2030, and it will double among African-Americans within this timeframe. To further highlight the direness, at present, one person develops Alzheimer disease every 72 seconds, and it is estimated that by 2050, one person will develop the disease every 33 seconds! Clearly, the sheer volume of new cases will create unprecedented burdens on our healthcare system and have a major impact on our economic system. As the most populous state, California will be disproportionately affected, stretching our public finances to their limits. To illustrate the economic impact of Alzheimer disease, studies show that an estimated $8.5 billion of care were provided in one year in the state of California alone (this value does not include other economic aspects of Alzheimer disease). Therefore, it is prudent and necessary to invest resources to try and develop strategies to delay, prevent, or treat Alzheimer disease now. California has taken the national lead in conducting stem cell research. Despite this, there has not been a significant effort to utilize the power of stem cell biology for Alzheimer disease. This proposal seeks to reverse this trend, as we have assembled a world class group of investigators throughout the State of California and in [REDACTED] to tackle the most significant and critical questions that arise in translating basic research on human stem cells into a clinical application for the treatment of Alzheimer disease. This proposal is based on an extensive body of preliminary data that attest to the feasibility of further exploring human stem cells as a treatment for Alzheimer disease.
Progress Report: 
  • Over the past decade, the potential for using stem cell transplantation as a therapy to treat neurological disorders and injury has been increasingly explored in animal models. Studies from our lab have shown that neural stem cell transplantation can improve cognitive deficits in mice resulting from extensive neuronal loss and protein aggregation, both hallmarks of Alzheimer’s Disease pathology. Our results support the justification for exploring the use of human derived stem cells for the treatment of Alzheimer’s patients.
  • During the past few months, we have begun studies aimed at taking human derived stem cells from the bench top to the bed side. To identify the best possible human stem cells to use in our future studies, we have conducted comparisons between a wide array of human stem cells and a mouse neural stem cell line (the same mouse stem cells used in the studies mentioned above). Using these results, we have selected a cohort of human stem cell candidates to which we will continue to study in upcoming experiments involving our AD model mice.
  • In addition to identifying the best human stem cells to conduct further studies, we have also performed experiments to determine the optimal immune suppression regimen to use in our human stem cell engraftment studies. Similar to organ transplants in humans, we will need to administer immune suppressants to mice which receive our candidate human stem cells. Our group has identified a potential suppressant, also found to work in humans, which we will use in future studies.
  • Over the past decade, the potential for using stem cell transplantation as a therapy to treat neurological disorders and injury has been increasingly explored in animal models. Studies from our lab have shown that neural stem cell transplantation can improve cognitive deficits in mice resulting from extensive neuronal loss and protein aggregation, both hallmarks of Alzheimer’s Disease pathology. Our results support the justification for exploring the use of human derived stem cells for the treatment of Alzheimer’s patients.
  • During the past few months, we have begun studies aimed at taking human derived stem cells from the bench top to the bed side. To identify the best possible human stem cells to use in our future studies, we have conducted comparisons between a wide array of human stem cells and a mouse neural stem cell line (the same mouse stem cells used in the studies mentioned above). Using these results, we have selected a cohort of human stem cell candidates to which we will continue to study in upcoming experiments involving our AD model mice.
  • In addition to identifying the best human stem cells to conduct further studies, we have also performed experiments to determine the optimal immune suppression regimen to use in our human stem cell engraftment studies. Similar to organ transplants in humans, we will need to administer immune suppressants to mice which receive our candidate human stem cells. Our group has identified a potential suppressant, also found to work in humans, which we will use in future studies.
  • During the last reporting period the lab has made substantial advancements in determining the effects of long term human neural stem cells engraftment on pathologies associated with the advancement of Alzheimer's disease. In addition, data obtained by our lab has may provide additional insight on ways to target the immune system as a means of prolonging neural stem cell survival and effectiveness.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-A
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 472 634
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
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.
Progress Report: 
  • In order for the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, 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.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This next-generation model system will provide a testing platform to evaluate the importance of matching immune system components known as human leukocyte antigens (HLAs). Because mouse and human immune systems are fundamentally different, these cutting-edge ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). 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 and to monitor for graft acceptance vs. rejection.
  • During this first year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 1: To establish mouse models with human immune systems (year 1). Firstly, we have increased purity of HSCs from 75% to 93%. This has enabled us to complete our second goal of generating 10 mice bearing 50% or more human immune cells. Thirdly, we have characterized the human adaptive immune systems of these mice and have found presence of 40-60% of human T lymphocytes in lymphoid organs of ‘humanized’ mice.
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). 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 and to monitor for graft acceptance vs. rejection.
  • During this second year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 2, which is designed to perform HLA haplotype ‘mix and match’ experiments using hES cell-derived NSCs as donors and ‘humanized’ mice as recipients (year 2). Firstly, we have now successfully generated ‘humanized’ mice that have 50% or more engraftment of human immune cells in lymphoid organs, defined as percentage of human immune cells within the mouse. Secondly, we have successfully HLA haplotyped these human donor CD34+ HSCs, and have additionally transplanted hES cell-derived NSCs with known HLA haplotypes. Finally, we have ‘mixed and matched’ HLA haplotypes in adoptive transfer experiments using human HSC reconstituted mice as recipients and human NSCs as donors. This critically important new tool will allow for a predictive model of human stem cell transplant acceptance vs. rejection.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-B
Investigator: 
Type: 
PI
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.
Progress Report: 
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). 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 and to monitor for graft acceptance vs. rejection.
  • During the third year of CIRM funding, we have addressed two specific questions that have arisen during the completion of Specific Aim 2: 1) which component of the HLA haplotype is most important to match in order to prevent brain stem cell rejection, and 2) can we expand blood stem cells obtained from a single umbilical cord blood sample? In response to question 1, we have determined that HLA-A is expressed at significantly higher levels in NSCs than the other HLA components, which makes this HLA type the critical player in immune system acceptance-rejection. As evidence of this, ‘humanized’ mice transplanted with NSCs expressing completely mismatched HLA-A elicited an immune response. Regarding question 2, we were able to accomplish ex vivo expansion of HSCs while maintaining their ‘stem-ness’ properties, which allows us to coordinate between the birth of mouse pups and the isolation of HSCs from umbilical cord blood samples, and also to significantly increase cell numbers to generate more ‘humanized’ mice. Additionally, in collaboration with Dr. George Liu from Cedars-Sinai Medical Center, we utilized ‘humanized’ mice to successfully model another disease that has become a threat to Californians’ health: skin infection by Staphylococcus aureus. While mice are generally not susceptible to this ‘human selective’ disease, ‘humanized’ mice did respond to the infection, closely mimicking the skin lesions observed in humans.
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). 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 and to monitor for graft acceptance vs. rejection.
  • During this no-cost extension (year 4) of CIRM funding, we have addressed both Specific Aims 2 and 3, and have specifically answered the following questions: 1) is the HLA-A haplotype important to match in order to prevent brain stem cell rejection, and 2) what are the transcriptome profiles of mouse vs. human compartments? In response to question 1, we have determined that HLA-A is expressed at significantly higher levels in NSCs than the other HLA components, which makes this HLA type the critical player in immune system acceptance-rejection. As evidence of this, ‘humanized’ mice transplanted with NSCs expressing completely mismatched HLA-A elicited an immune response. Regarding question 2, we were able to accomplish a new technique utilizing RNA sequencing technology on brain sections from 'humanized' mice engrafted with human NSCs. Additionally, in collaboration with Dr. George Liu from Cedars-Sinai Medical Center, we utilized ‘humanized’ mice to successfully model another disease that has become a threat to Californians’ health: skin infection by Staphylococcus aureus. While mice are generally not susceptible to this ‘human selective’ disease, ‘humanized’ mice did respond to the infection, closely mimicking the skin lesions observed in humans. A manuscript has recently been submitted detailing this work to the Journal of Experimental Medicine.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01720
Investigator: 
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.
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01107
Investigator: 
Name: 
Type: 
PI
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.
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$918 000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells have considerable potential as sources of differentiated cells for numerous biomedical applications. The ability to introduce targeted changes into the DNA of these cells – a process known as gene targeting – would have very broad implications. For example, mutations could readily be introduced into genes to study their roles in stem cell propagation and differentiation, to analyze mechanisms of human disease, and to develop disease models to aid in creating new therapies. Unfortunately, gene targeting efficiency in hESCs is very low. To meet this urgent need, we propose to develop new molecular tools and novel technologies for high efficiency gene targeting in hES and iPS cells. Importantly, this approach will be coupled with genome-wide identification and functional analysis of genes involved in the process in dopaminergic neuron development, work with fundamental implications for Parkinson's disease. Barriers to targeted genetic modification include the effective delivery of gene targeting constructs into cells and the introduction of defined changes into the genome. We have developed a high throughput approach to engineer novel properties into a highly promising, safe, and clinically relevant gene delivery vehicle. For example, we have engineered variants of this vehicle with highly efficient gene delivery to neural stem cells (NSCs), and the resulting vehicles can mediate efficient gene targeting. We now propose to engineer novel gene delivery and targeting vehicles optimized for use in hESCs and iPS cells. One application of such an improved vector system will be to study the mechanism of ESC differentiation into dopaminergic neurons aided by the key transcription factor Lmx1a. We propose to identify target genes that are regulated by Lmx1a during dopaminergic neuron differentiation using the newly developed technique of ChIP-seq, in combination with RNA expression and bioinformatics analysis. This work will identify essential control genes that drive dopaminergic neuron differentiation. Furthermore, our improved gene delivery and targeting system will be used for overexpressing candidate genes, knocking them down via RNA interference, and knocking in reporter genes to analyze gene expression networks during neuronal differentiation. The generation of efficient targeting technologies, in combination with genome wide analysis of gene regulation networks, will provide a general method for identifying and testing key regulatory genes for stem cell self-renewal and differentiation, as well as generating stem cell-based models of human disease. This blend of bioengineering and cell biology therefore has strong potential to create an important new capability for basic and applied stem cell research.
Statement of Benefit to California: 
This proposal will develop novel molecular tools and methodologies 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 introducing specific genetic modifications into a stem cell genome is a greatly enabling technology that would impact many downstream medical applications. This capability will further enable investigations of self-renewal and differentiation, two defining properties of human stem cells. New tools to introduce targeted alterations of ES and iPS cells will also yield key model systems to elucidate mechanisms of human disease, and most importantly enable the generation of mutant cell lines to serve as models of human disease and systems for high throughput screening to develop novel therapies. Finally, the reverse process, the repair of genetic lesions responsible for disease, can in the long run enable the generation of patent-specific stem cell lines for therapeutic application. Each of these applications will directly benefit biomedical knowledge and human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and utilization of efficient homologous recombination techniques for gene targeting in human stem cells, the development of safer and more effective viral vectors for gene transduction in human stem cells, and the development and analysis of human embryonic stem cell lines with reporter genes inserted into key loci – 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: elucidating mechanisms of ES cell differentiation into dopaminergic neurons, part of a critical path towards developing therapies for Parkinson’s disease. While hESCs clearly have this capacity, the underlying mechanisms are incompletely understood, and the efficiency of this process must be improved. We will elucidate transcriptional networks that underlie this process, and utilize our novel gene targeting system to identify and analyze key components of these networks. This work will lead to a better fundamental understanding of mechanisms regulating stem cell differentiation, as well as enhance our ability to control this complex process for biomedical application. The co-investigators have a strong record of translating basic science and engineering into practice through interactions with industry, including the founding of biotech companies in 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: 
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.
Funding Type: 
Shared Labs
Grant Number: 
CL1-00501-1.2
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$5 893 682
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation. Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body. In this proposal, we will build out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. We seek to understand how hESCs differentiate into authentic, clinically useful nerve cells and will use novel molecular tools to examine the behavior of cells transplanted in animal models of human neurological disease. We will also need to develop a noninvasive method of following cells after transplantation and we propose to develop luciferase-tagged (light-emitting) hESC lines for in vivo animal imaging. In addition, we will use hESC-derived nerve cells to screen drug and chemical libraries for compounds that protect nerve cells from toxicity, and to develop in vitro disease models. We believe that these experiments are critical to enhancing our understanding of neurological diseases and providing the tools that will be necessary to move cell therapy to the clinic. Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We propose to teach four 5-day hands-on training courses – two basic and two advanced hESC culture courses per year – to California scientists free of charge. These courses will provide scientists with an understanding of hESC biology and will enable them to set up and conduct hESC research after completion of training. In summary, the goal of this proposal is to provide over twenty investigators at the home institute and neighboring institutions with the ability to culture, differentiate, and genetically manipulate hESCs – including clinical-grade hESC lines – to develop diagnostic and therapeutic tools.
Statement of Benefit to California: 
We propose to build a Shared Research Laboratory and offer a Stem Cell Techniques Course for over twenty principal investigators at the home institute and neighboring institutes working collaboratively on stem-cell biology and neurological diseases of aging. We propose to: 1) Purify nerve cells at different stages of maturation from human embryonic stem cells and to develop transplantation strategies in animal models that mimic human diseases, including Parkinson’s disease, stroke and spinal cord injuries; 2) Screen drug and chemical libraries for reagents that protect nerve cells from toxicity and develop in vitro disease models using nerve cells generated from human embryonic stem cells; and 3) Assess the long-term integration and differentiation of transplanted cells using a non-invasive imaging system. We believe these experiments provide not only a blueprint for moving stem-cell transplantation for Parkinson’s disease toward the clinic, but also a generalized plan for how stem-cell therapy can be developed to treat disorders like motor neuron disease (amyotrophic lateral sclerosis, or Lou Gehrig’s disease) and spinal cord injury. As the only stem-cell research facility in California’s 10-12 most northwest counties, we are uniquely positioned to extend the promised benefits of Proposition 71 to this large part of the state. The tools and reagents we develop will be made widely available to California researchers and we will select California-based companies for commercialization of any therapies that may result. We also hope that California-based physicians will be at the forefront of translating this promising avenue of research into clinical applications. Finally, we expect that the money expended on this research will benefit the California research and business communities, and that the tools and reagents we develop will help accelerate stem-cell research in California and worldwide.
Progress Report: 
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation. Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. Supported by this facility, we have in the past year successfully developed a process for the production of functional dopaminergic neurons from hESCs that are suitable for potential clinical uses, e.g., in treating Parkinson’s disease (Parkinson’s disease is caused by the death of dopaminergic neurons). Our system provides a path to a scalable Good Manufacture Practice (GMP)-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential drug screening applications. In addition, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in GMP protocol that can be safely used to deplete unwanted contaminating precursor cells from dopaminergic neurons, a target for cell therapy.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 7 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation.
  • Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body. In this proposal, we will build out of approximately 3800 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 420 square feet for classroom facilities dedicated to training in hESC culture and manipulation. We seek to understand how hESCs differentiate into authentic, clinically useful nerve cells and will use novel molecular tools to examine the behavior of cells transplanted in rodent models of human neurological disease. We will also need to develop a noninvasive method of following cells after transplantation and we propose to develop luciferase-tagged (light-emitting) hESC lines for in vivo animal imaging. In addition, we will use hESC-derived nerve cells to screen drug and chemical libraries for compounds that protect nerve cells from toxicity, and to develop in vitro disease models. We believe that these experiments are critical to enhancing our understanding of neurological diseases and providing the tools that will be necessary to move cell therapy to the clinic.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We propose to teach four 5-day hands-on training courses: two basic and two advanced hESC culture courses per year, to California scientists free of charge. These courses will provide scientists with an understanding of hESC biology and will enable them to set up and conduct hESC research after completion of training.
  • In summary, the goal of this proposal is to provide over twenty investigators at the home institute and neighboring institutions with the ability to culture, differentiate, and genetically manipulate hESCs - including clinical-grad hESC lines—to develop diagnostic and therapeutic tools.

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