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RL1-00677-1: Derivation of new induced pluripotent stem (iPS) cells to build neurological disease models

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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

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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None