Funding opportunities

Generating Pluripotent Stem Cells from Skin of Patients with Multiple Sclerosis and Parkinson's Disease

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00683
Funds requested: 
$1 479 600
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The goal of this proposal is to directly derive patient-specific induced pluripotent stem cell (iPSC) lines from skin of patients with Multiple Sclerosis (MS) and Pakinson’s Disease (PD). A patient-specific iPSC line is a virtual clone of the patient because most cells of that patient can be differentiated from his/her iPSC in laboratory. Once they become available, those iPSC lines will allow scientists in any laboratory to obtain a large number of various types of cells from those MS and PD patients via in vitro differentiation. One obstacle for understanding disease mechanisms is the lack of in vitro systems to study the development of disease, especially for diseases of the human central nervous system (CNS) such as MS and PD. Animal models of the CNS diseases often fail to duplicate the true human diseases. Patient-specific iPSC generated by our proposal have the potential to provide all scientists unlimited access to diseased cells of individual patients. We have constructed lentivirus that can introduce a stem cell selection marker into human skin cells with high efficiency and stability. We also successfully reprogrammed skin cells into iPSC in our laboratory. The high efficiency stem cell selection marker allows the selection of rare successfully reprogrammed cells. With access to one of the largest MS and PD patient populations in California, we are able to obtain skin biopsies from patients with various genetic background and pathological status. Acknowledging the value of MS iPSC and our expertise, the National MS Society has awarded us a grant to begin the work of establishing MS iPSC. Because non-NIH approved human embryonic stem cell (hESC) lines will be used in our cell fusion experiment, federal funding can not be used in our proposal. With our MS society funded proposal as a starting point, we propose to generate a collection of disease-specific iPSC lines from MS and PD patients. We will first reprogram our MS skin cells into iPSC, which have a stem cell selection marker introduced by lentivirus. Second, PD iPSC lines will be generated with similar procedures. Finally, a collection of iPSC lines will be generated from patients with diverse genetic background and pathological status of MS and PD. Besides distributing those MS/PD iPSC to the research community, our proposal develops a protocol for large-scale systematic generation of patient-specific iPSC collections of various diseases, which impacts the whole stem cell field beyond MS and PD.
Statement of Benefit to California: 
Approximately one of every thousand Californians suffers from multiple sclerosis (MS), which is the most common neural disorders without known cause, means of prevention, or cure. Parkinson’s Disease (PD) is the second common neurodegenerative disorder and affect one in 100 individuals over the age of 50. Here, we propose to derive induced pluripotent stem cell (iPSC) lines from skin of MS and PD patients. These cells can be used to identify genes and pathways that are involved in MS and PD as well as for drug discovery for treating MS and PD. These iPSC will directly benefit the large population of MS and PD patients in California. The protocol for generating disease-specific stem cell lines developed in this proposal will impact the whole field of stem cell research and application.
Review Summary: 
Executive Summary This is a proposal to generate 10 induced pluripotent stem (iPS) cell lines from patients with Multiple Sclerosis (MS) or Parkinson’s disease (PD). The iPS cells will be generated from skin biopsy fibroblasts by established methods of lentiviral introduction of pluripotency inducing transcription factors. The idea is that these lines will be valuable to those interested in MS and PD, for applications such as drug testing. Reliable human cell-based models of MS and PD would provide valuable tools to study the cellular and molecular mechanisms of these diseases, and could lead to new therapies. However, reviewers felt that the proposal lacks cohesiveness by concentrating on two very different diseases and criticized the lack of attention to the complexity of the two diseases. A major concern with this proposal was the lack of detail regarding the disease models chosen. In MS, the overall consensus is that this is an acquired disease of autoimmune origin in which immune cells attack myelin and oligodendrocytes with associated loss of axons. What one can learn from iPS cell lines from MS patients should have been addressed in more detail by the applicant. The only note about how cells from these patients might be used is the mention of their differentiation into oligodendrocytes. However, no reliable protocols for oligodendrocyte differentiation currently exist, and importantly, a reviewer questioned the utility of studying neural cells in this context since MS is an autoimmune disease. Overall the rationale for the proposed experiments is weak. A similar concern was voiced with the choice of Parkinson’s disease. The majority of patients have a late onset idiopathic form of the disease. There was no discussion of the genetics or clinical heterogeneity of PD, and no note was made of whether the applicant will select patients with familial forms of the disease. Reviewers argued that cell lines will be derived from such differing pathological backgrounds that it might be hard to use them to define general mechanisms. Thus the utility of cell lines representing these two diseases may be questionable and should have been discussed in more depth. Reviewers agreed with the applicant that a better method for selecting re-programmed cells is required, especially in a project such as this one where multiple lines are to be created from primary skin biopsies. However, the method they propose to use is not backed up by any preliminary data, calling the feasibility of the application into question. Moreover, the introduction of the indicated marker gene into patients’ fibroblasts to serve as a reporter of successful reprogramming is unnecessary given previously published results, and may even complicate understanding disease pathology by introducing yet another randomly inserted piece of exogenous DNA into the genome. In fact, no attention was paid to the effects of random sites of gene insertion of the multiple transcription factors. Given that there will be many such insertions in each cell line; this could have a major effect on the behavior and utility of the cells. Another important point raised by reviewers was the fact that given the costs requested, it would have been desirable to have seen at least some thought given to specific applications for the cells, e.g. an initial analysis of their specific neural differentiation capacity, clearly essential if they are to have utility for drug discovery. Reviewer One Comments Significance: Cell lines from patients with specific diseases are an obvious and valuable use of the iPS technology. However, this project raises two questions. First, as the cell lines will be generated from patients with no apparent selection for disease type, genotype etc., the variability of the disease becomes a real issue. Would it be better to focus in the initial studies on a well-defined subgroup of MS or PD patients, e.g. in PD those with specific genetic mutations causing the disease? My concern is that without this selection the value of the product here is limited. While this study will certainly show that it is possible to generate patient specific iPS cells they will be from such differing pathological backgrounds that it might be hard to use them to define general mechanisms. Second, no attention in the application is paid to the effects of random sites of gene insertion of the multiple transcriptional factors. Given that there will be many such insertions in each cell line this could have a major effect on the behavior and utility of the cells. Again, this does not negate the value of these early studies, but it does require consideration. Feasibility: The authors make an excellent point in this proposal that a better method of selecting re-programmed cells is required, especially in a project such as this one where multiple lines are to be created. While consideration of this is a strength in the present application, the method they propose (viral infection of fibroblast with a Rex-gfp construct that is activated in stem cells as it is a downstream target of Oct4) is unfortunately not backed up by any preliminary data. As this is a key part of their experimental plan, it must raise questions as to feasibility. Also, given the costs requested it would have been desirable to have seen at least some thought given to specific applications for the cells, e.g. an initial analysis of their specific neural differentiation capacity, clearly essential if they are to have utility for drug discovery Responsiveness to RFA: Pluripotency is likely, but testing is not described in the research design (Aim 5 is missing). However, as I have said above, the analysis proposed in the summary is limited and this will certainly limit the value of these cells to the community. Reviewer Two Comments Significance: The goal of the proposal is to generate iPS cell lines from skin biopsies (primary fibroblasts) of patients with multiple sclerosis (MS) and Parkinson’s disease. They will then make these cell lines available to scientists who wish to study the disease in vitro. My major concern with this proposal is with the two diseases chosen. In MS, the overall consensus is that this is an acquired disease of autoimmune origin in which there is an attack against myelin and oligodendrocytes with associated loss of axons. What one can learn from iPS cell lines from MS patients escapes me or should have been addressed in more detail by the applicant. The only note about how cells from these patients might be used is the mention of their differentiation into oligodendrocytes. However the current success in differentiating these cells from neural stem cells derived from hES cells is dismal with the exception of one laboratory. This does not sound like a promising venture for iPS cells at the moment! Likewise with the choice of Parkinson’s disease. The majority of patients have idiopathic disease of late-stage origin. No note was made of whether they will select patients with familial forms of the disease. Thus the utility of cell lines from these two diseases may be questionable and should have been discussed in more depth. Feasibility: The experimental design is quite carefully documented with a step-by-step approach toward generating iPS cells. They present supportive data of their ability to culture fibroblasts (presumably human though not stated) and early evidence of reprogramming of mouse fibroblasts. The applicant is an Assistant Professor at USC in Pathology with relatively modest current funding and limited publication list. He does have a pilot grant from the NMSS to initiate the work on establishing cell lines form MS patients. He has appropriate clinical connections as the source for the skin biopsies. He has access to the core stem cell facilities at USC. Responsiveness to RFA: Appropriate testing of the pluripotency of the cells is planned and sharing them is a major goal of the applicant. Reviewer Three Comments Significance: The goal of this proposal is to generate induced pluripotent stem (iPS) cells from patient fibroblasts with Multiple Sclerosis (MS) and Parkinson’s disease (PD). Reliable human cell-based models of MS and PD would provide tools to study the cellular and molecular mechanisms of these diseases and could lead to new therapies. Feasibility: This proposal has five Aims, two of which (Aims 2 and 3) are not well justified and the logic for there inclusion unclear. The introduction of an hREX-GFP reporter into patient fibroblasts to serve as a reporter of successful reprogramming is unnecessary given previously published results and may even complicate understanding disease pathology by introducing yet another randomly inserted piece of exogenous DNA into the genome. The proposal lacks cohesiveness by concentrating on two very different diseases MS and PD. There is very little detail given about whether they would focus on known familial forms of the diseases or sporadic forms and by only proposing to create 10 cell lines for these very broad and varied diseases they run the risk of having very different cellular and molecular pathologies that may not help uncover disease mechanism. Overall the logic and justification for creating lines from MS patients, an autoimmune disorder is also rather vague. Responsiveness to RFA: The proposal is very responsive to the RFA.
Conflicts: 

© 2013 California Institute for Regenerative Medicine