Funding opportunities

Derivation of new induced pluripotent stem (iPS) cells to build neurological disease models

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00677
Funds requested: 
$1 374 192
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Embryonic stem cells (ESCs) hold great potential for cell replacement therapies, in which the cells are lost due to disease or injury, since they are capable of proliferating perpetually and differentiating into multiple cell types depending on environmental and genetic factors. Despite the therapeutic potential of human embryonic stem cells (hESCs), clinical applications could be limited because of immune rejection. In addition, the use of hESCs has been the central focus of many ethical and political discussions. Therefore, many scientists, including us, have been searching for alternative sources for pluripotent stem cells. Recently, it has been shown that pluripotent stem cells (referred to as "iPS cells) can be obtained from somatic cells such as fibroblasts. The generation of iPS cells has created significant amount of excitement in the stem cell biology field, since it offers the possibility of creating stem cells from the somatic cells of the same individual. This approach not only circumvents the immune rejection problem but also the ethical concerns. However, it is not yet known whether iPS cells behave like hESCs functionally. Furthermore, differentiation of iPS cells into the cells of the central nervous system, such as neurons or glia, has not been demonstrated. In this application, we propose to derive patient-specific iPS cells from fibroblasts using alternative methods, to improve quality, safety, and test functionality while establishing in vitro models to study underlying molecular mechanisms of neurological diseases. Our laboratory has extensive expertise in handling hESCs. We created a highly efficient method to differentiate hESCs into neurons or glia. First, we will compare the functionality of iPS cells and hESCs. Using our established protocol, we will subsequently differentiate iPS cells into neurons. The characteristics of iPS cell-derived neurons will be documented using numerous techniques. Following the successful generation and characterization of iPS cell-derived neurons, we will begin to generate disease-specific iPS cells. We will collect dermal samples from Parkinson, Huntington and Rett Syndrome patients, since all three conditions have defined genetic alterations. The results of these experiments will allow us to use iPS cell-generated neurons as models to study underlying molecular mechanisms of neurological diseases. Ultimately, we will use these disease models to begin developing therapeutic approaches.
Statement of Benefit to California: 
Despite the therapeutic potential of human embryonic stem cells (hESCs), clinical applications could be limited because of immune rejection and ethical concerns. Recently, it has been shown that pluripotent stem cells (referred to as "iPS cells) can be obtained from somatic cells such as fibroblasts as an alternative source of pluripotent stem cells. This approach not only circumvents the immune rejection problem but also the ethical concerns. However, it is not yet known whether iPS cells behave like hESCs functionally. In this application, we propose to derive disease-specific iPS cells from fibroblasts taken from patients with Parkinson's disease, Huntington's disease or Rett Syndrome. The results of these experiments will allow us to use iPS cell-generated neurons as models to study underlying molecular mechanisms of neurological diseases. Ultimately, we will use these disease models to begin developing therapeutic approaches. Many Californians suffer from diseases that can possibly be cured by using stem cell technology. In this proposal, we designed experiments to create new stem cells using skin cells (i.e., fibroblasts). With this approach, we can study the underlying mechanism of many neurological disorders, such as Parkinson's disease (PD). PD is the second leading neurodegenerative disease, and there is no cure currently available. In comparison to other states in the U.S., California is among one of the states with the highest incidence of PD. The genetic component of the PD has been established. Using the proposed models of iPS cells of this application, we will begin to understand the etiology of PD. Better understanding of the genetics of PD will assist us to reveal how environmental factors participate in the development of PD. California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the U.S. (Cal Pesticide use reporting system). A commonly used herbicide, paraquat, has been shown to induce parkinsonism in both animals and human. It has been shown that the incidence of PD-caused mortality was higher in counties where agricultural pesticide are used in comparison to the counties that do not use pesticides in California. Furthermore, California has the largest Hispanic population in the U.S. Studies suggest that incidence of PD is the highest among Hispanics (Van Den Eeden et al, American Journal of Epidemiology, Vol. 157, pages 1015-1022, 2003). Thus, finding effective treatments for neurodegenerative and genetic disorders, such as -but not limited to- PD, will significantly benefit the citizens of California.
Review Summary: 
Executive Summary This proposal aims to develop novel methods to derive induced pluripotent stem (iPS) cells from fibroblasts, and to use these methods to derive neurons from patients with neurological diseases in order to establish in vitro disease models. In Specific Aim 1, the applicant will attempt to generate iPS cells using adenoviral infection or electroporation of the genes already known to reprogram adult cells. In Aim 2, the applicant plans to differentiate the iPS cells to a neuronal lineage via embryoid bodies. Resulting neurons will be analyzed at multiple levels to determine their function in vitro and in vivo after transplantation. In Aim 3, patient-specific iPS cells will be generated from patients with Parkinson’s disease (PD), Huntington’s disease (HD), and Rett Syndrome. The resulting iPS cell-generated neurons will be used as in vitro models to study underlying molecular mechanisms of neurological diseases that are essential for the development of therapeutic approaches. The questions to be asked in this proposal are straightforward and include some of the most significant problems associated with the potential use of iPS cells using current technology. Reviewers described three main points that provide support in terms of significance for this proposal. 1) The realization that non-integrative methods are needed to improve iPS cell generation. 2) The plan to convert iPS cells into neurons and examine their properties 3) The plan to generate disease-specific cell lines. Despite the potential significance of the proposal, reviewers criticized that it was unfocused and overly ambitious. The project suffers by having two ambitious aims; to develop new methods to generate iPS cells and to develop disease models for several complex neurological disorders. Furthermore, each of the proposed aims was weakened by conceptual concerns which decreased reviewers’ enthusiasm for the proposal. In Specific Aim 1, the approach will be to create pluripotent cells using non-integrative genetic approaches. A major criticism of these experiments was that adenovirus, the proposed alternative, is also a virus and might have the same problems as the retroviral system. Reviewers were thus concerned about the long term utility of the method, although they were more positive about the proposed testing of electroporation as an alternative non-integrative genetic approach. The applicant will then compare iPS cells generated by established methods and by the new methods proposed in this application with bona fide embryonic stem (ES) cells using a battery of markers. Reviewers raised concern that the use of only three transcription factors for re-programming means that the question of pluripotency needs to be considered more carefully than proposed. This application does not deal with this in a thorough way,e.g. there is no mention of testing the pluripotency of the cells by teratoma formation or other methods. In the second Specific Aim, the goal is to differentiate the iPS cells into functional neurons. Although one reviewer thought that analysis of mitochondrial function as cells differentiate into neurons was interesting, to another the reason for this focus was unclear and left him/her wondering whether the choice of experiments reflected the expertise of the investigators rather than any biological imperative. Moreover, critical transplantation experiments are very poorly explained. In the third Aim the applicants plan to generate iPS cells from skin biopsies from patients with neurological diseases, and to use them to develop neurons in culture. Reviewers found the generation of patient-specific lines to be the most original feature of the application, but had several concerns with the project proposed. First, they noted that the choice of diseases was very broad. This was considered a problem as the applicant is trying to tackle three neurological diseases that are very heterogeneous in terms of their cellular and molecular pathological mechanisms. Second, several important aspects related to the diseases in question were not discussed in the application. In the case of HD patients, for instance, definition of the patients’ specific molecular defect, i.e. their CAG expansion size, is necessary. Rett syndrome is X-linked, a fact that would impact the study but is not discussed at all in the application. Finally, in the case of PD, it is not clear whether the patient(s) sampled will be those rare cases with a genetic basis of the disease. Another concern raised by reviewers was that the applicant did not provide evidence of access to patients with neurological diseases (in order to obtain the clinical samples). In general the project seems feasible given the prior work of others and the published expertise of the authors in neuronal differentiation and ES cell biology. In terms of responsiveness to the RFA, the review panel found that the cells generated will have a value. However, the disease-specific cell lines will only be useful if more attention is paid to the documentation of the patients’ clinical state and to the experimental methodologies (e.g. obtaining cells from unaffected relatives) to be able to explore the role of genetic background on the expression of the disease. Reviewer One Comments Significance: The three significant factors of this application are: 1) The realization that non integrative methods are required for iPS cell generation. 2) The plan to convert iPS cells into neurons and examine their properties 3) The plan to generate disease specific cell lines. That said, the focus for iPS cell generation is still on viral methods (adenoviruses) which will raise concerns over long term utility. The analysis of neuronal function focuses on mitochondria, for reasons that are not really explained and probably reflect the expertise of the investigators rather than any biological imperative. The critical transplantation experiments in particular are very poorly explained. The patient specific lines are the most original feature of the application, but no attention has been paid to the issue of disease heterogeneity. Feasibility: In general the project seems feasible given the prior work of others and the published expertise of the authors in neuronal differentiation and ES cell biology. However, there are a few areas of concern. 1) Why are only three transcription factors to be used for the re-programming? I see the justification for not using cMyc, but no convincing evidence is presented in the preliminary data that these three factors will be sufficient to create pluripotent lines. We are shown only morphology and expression of a few stem cell markers. 2) More preliminary data on neuronal function of cells differentiated from the embryonic stem (ES) cell lines is required. For example, do human embryonic stem cell (hESC) derived neurons integrate following transplantation, which could be analyzed by function and/or morphology. How will cell fusion be excluded? 3) Given the recognized need to avoid viral vectors if at all possible, I was surprised that more attention was not paid to the use of the Amaxa (electroporation) technology. 4) For the clinical samples required, where is the evidence that the applicants have access to these patients? In the HD patients, how will expansion size be defined? Rett’s is an X-linked condition – how will the state of X inactivation (which will very likely be skewed) in the cells be defined? Responsiveness to RFA: The cells generated will have a value but, in the case of those derived from normal human fibroblasts, the use of only three transcription factors for re-programming means that the question of pluripotency needs to be considered very carefully. This application does not deal with this in a thorough way. The clinical cell lines will only be useful if more attention is paid to the documentation of the patients’ clinical state and methodologies (e.g. obtaining cells from unaffected relatives) to explore the role of genetic background on the expression of the disease Reviewer Two Comments Significance: The questions to be asked in this proposal are straightforward and include some of the most significant problems in the potential use of iPS cells using the technology as currently described. These are, the use of retroviruses as a means of transducing the cells, the determination that iPS cells have a similar phenotype to hES cells, and whether the differentiated progeny, in the case of neurons, is functional. The approaches described to accomplish this are probably being used by a number of labs worldwide and so are possibly not entirely novel. Nonetheless, these are some of the key points that require answering. It will be a tight race to be the first to answer them however! Likewise, producing neuronal cell lines in this fashion from specific disease may not be entirely novel yet it is one of the major goals of hES cell research. Feasibility: Again, the design of the proposed experiments seems straightforward. In Specific Aim 1, the approach will be to create pluripotent cells using non-integrative genetic approaches. Human fibroblasts will be purchased and then transfected with adenovirus encoding for Oct4, Sox2 and Klf4. As an alternative plan, they will use electroporation. They will compare these results with retrovirally transduced cells. Once generated, a battery of markers will be used to determine their relationship to bona fide ES cells. If the efficiency of transformation with adenovirus is low, they will use a retrovirus but notes that c-myc will not be included. This however still leaves open the question of retroviral-induced problems. No note is made here however of testing the pluripotency of the cells i.e. teratoma formation etc. In the second Specific Aim, the goal is to differentiate the iPS cells into functional neurons and this will be evaluated thoroughly in multiple ways. Interestingly and perhaps somewhat novel, they will characterize mitochondrial structure and function as the cells differentiate into neurons. They hypothesize that the functional state of mitochondria controls iPS production and their differentiation to neurons. The proposed analysis of mitochondria is detailed and will possibly yield novel information. To differentiate cells toward neuronal lineage they will use protocols already used in their lab on hESCs. A battery of neuronal and glial markers, as well as neuronal sub-type markers will be examined for. They will compare the epigenetic changes that occur during iPS induction from fibroblasts and then differentiation into neurons. It is not entirely clear what this information will be used for. They have established an important collaboration with T. Sudhoff who will examine the function of derived neurons physiologically. He will document the formation of functional synapses and the sub-type of neurons investigated. To complete functional analyses, they will transplant iPS cell-derived neural progenitors into the brain of SCID mice. In the third Aim they use iPS cells derived from skin biopsies of patients with neurologic disease, to develop neurons in culture. For Parkinson’s disease they are particularly interested in driving cells to dopaminergic neurons. It is not clear here however whether in this case the patient/s sampled will be those rare cases with a genetic basis of the disease. They mention this but not that the biopsies will be from such cases. The applicant is well qualified to perform the proposed studies. She is now an Associate Professor at UCLA, having spent five years at Harvard in M. Greenberg’s lab. Her publication record in the field is good, and her funding is quite good. The preliminary data she presents confirms her technical abilities with hESCs and ability to induce neuronal differentiation. She also provides evidence that she can induce reprogramming in adult mouse fibroblasts with preliminary studies ongoing to derive iPS cells from human fibroblasts. She has all the space and equipment needed to do the work. Important collaborations have been established with M. Teitell a fellow faculty member at UCLA (mitochondrial evaluation) and with Thomas Sudhoff (UT-Southwestern). Although there are no letters of collaboration from either, she has a recent PNAS paper (2007) with the latter. Responsiveness to RFA: Yes it is likely that the cells derived will be pluripotent but limited testing of this is noted. It would also appear that all iPS cell lines generated will be available to all those who request them. Reviewer Three Comments Significance: The goal of this proposal is to create induced pluripotent stem (iPS) cell lines from fibroblasts taken from patients with Parkinson’s disease (PD), Huntington’s disease (HD), and Rett Syndrome (RS). These diseases impose a significant health care burden upon California and the US. Reliable human cell-based models for these diseases promise to help uncover the molecular mechanisms responsible for the pathobiology of these diseases as well as provide a platform for discovering novel therapeutics. Feasibility: This proposal is clear and consistent in its design and objectives. The groups involved obviously have the expertise required to direct the differentiation of iPS cells into neuronal cell types and execute the analysis of disease phenotypes associated with PD, HD, and RS. The proposal suffers from a lack of focus, by choosing to address three diseases with very different cellular and molecular pathologies. The major flaw of the proposal is the reliance on untested non-integrative methods for generating iPS cells. Without preliminary data demonstrating robust sustained expression from these techniques the likelihood of creating iPS cells is low. Responsiveness to RFA: The proposal is very responsive to the RFA
Conflicts: 

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