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RL1-00641-1: Generation and Testing of Induced Pluripotent Stem Cell Lines for Understanding Human Oligodendrocyte Development and Disease

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Many human neurological disorders, as diverse as inherited leukodystrophies, periventricular leucomalacia, cerebral palsy, and multiple sclerosis, are characterized by the selective loss of oligodendrocytes, the myelin-forming cells in the central nervous system. In affected patients, an appropriate way to restore function may be to provide them with the relevant replacement cells, the oligodendrocytes. Scientists have already been working on methods to generate replacement oligodendrocytes from human embryonic stem cells (hESCs) or to generate accurate in vitro models of these human neurological diseases using hESCs. A recent breakthrough in stem cell research is the success of generating induced pluripotent stem cells (iPS cells) by in vitro reprogramming of human somatic cells back to a pluripotent state using defined factors. We will build upon this amazing technology to generate new iPS cells directly from adult cells from patients with myelin disorders either by using defined genetic transcription factors or by using small-molecule drugs/defined soluble factors, and test the utilities of these new cells in experimental models in the lab. We will compare the behaviors of the oligodendrocytes derived from these iP cells versus hESCs. Oligodendrocytes are the cell type that is diseased in patients with myelin disorders, so it would be ideal to derive iP cells directly from human oligodendrocytes (“dedifferentiation”) and then to “redifferentiate” them into better in vitro models to study oligodendroglial diseases. Such a “de- and re-differentiation” (hence making a complete “Cellular U-Turn”) approach represents an ideal model of “disease in a dish". Currently very few treatments for any neurological diseases exist, in part because of the lack of suitable in vitro models with which to test therapeutics. The proposal will lead to generation of new cell lines that have important research and clinical application. In vitro reprogramming could provide a way to generate patient-specific stem cells that, in culture, could be turned into the type of cell or tissue needed to cure the patient’s disease or injury. Moreover, genetic defects can be repaired in reprogrammed cells before being transplanted back into the patient’s body. Although both hESC or iPS cell-based therapeutic strategies that can promote remyelination will likely have significant clinical impact, one important advantage of patient-specific self-transplants is that they obviate the need for immunosuppression.

Statement of Benefit to California (provided by applicant)

We plan to develop methods to make a complete “Cellular U-Turn” involving “dedifferentiation and redifferentiation” of specialized cell types to study “disease in a dish”. In addition to being integral to understanding the mechanism by which the stem cell program can be reinstated in specialized cells, if successful, this work will provide an important milestone for regenerative medicine. The significance of accomplishing this work is thus at the highest possible level currently for the field. The proposal addresses fundamental biological processes in reprogramming, i.e. reverting specialized cell types back to a more immature cell that can act like a stem cell, and may also pave the way for autologous stem cell-based therapies. We believe the proposed research will benefit Californian in many ways. It will result in development of novel technologies that will be broadly applicable to study stem cells and development of stem cell-based therapies, and will help position us and other Californian scientists at the forefront of stem cell research and medicine. We will make newly generated cell lines readily and freely available to other investigators and companies in the hope of accelerating the pace of discovery. The research relies on products and tools manufactured and sold in the state of California. If successful, research will require a scaled-up version of protocols designed in the proposed studies. This could attract new biotechnology companies in the state, boosting the tax revenue in the state. This in turn will provide new jobs for California residents. This research will increase experience and knowledge of stem cells among residents of California. Establishment of successful cellular therapeutics in California will encourage institutions of higher education to promote science education to fill the jobs created by stem cell research. This will retain California students in the state that are interested in biomedical research and medical careers. It could also attract out-of-state students seeking degrees that will allow them access to careers in stem cell research. This research will contribute to the California education and health care systems by training undergraduate, graduate and postdoctoral students into highly skilled stem cell biologists. It is also envisioned that this will trickle down to the K-12 levels and provide funding to promote science education at all levels.

Review

This grant focuses on diseases of the oligodendrocyte, the myelin-forming cells of the central nervous system. The rationale behind this project is to take oligodendrocytes, reprogram them into induced pluripotent stem (iPS) cells, and then redifferentiate them, a process the applicant refers to as a cellular u-turn. Three sets of experiments are proposed: 1) Fibroblasts from patients with a specific form of leukodystrophy will be reprogrammed using existing technologies for producing iPS cells. These cell lines will then be compared with existing human embryonic stem cells (hESCs). 2) Normal human oligodendrocytes (obtained commercially) will be reprogrammed using viruses to express the transcription factors. Additionally, the effect of a previously identified small molecule, used in conjunction with growth factors known to inhibit oligodendrocyte differentiation, will be tested for their reprogramming activity. 3) These iPS cells will be differentiated into oligodendrocytes using an embryoid body-based approach already established in the lab, and tested in mouse models of periventricular leukomalacia. Cells will be transplanted either into the subventricular zone (SVZ) or intravenously, and their differentiation, survival, axon growth, behavior and migration will be assessed using magnetic resonance imaging (MRI)-detectable markers. The applicant proposes to use the reprogramming technology to develop a number of cell lines that would advance our understanding of glial diseases of the central nervous system.

Stem cells derived from patients with inherited myelin diseases such as the leukodystrophies will be a very valuable resource and represent an excellent way forward for studies of the pathogenesis of these diseases. Although there is great merit in the target and the application is put forward by an impressive investigator, the reviewers disagreed with the applicant regarding the value of using a cellular u-turn method. In addition, they felt that the proposal suffers from a lack of defined focus in terms of the patient population and the relevant animal model to study cellular therapy. The applicant has begun to develop protocols for generating oligodendrocytes and for studying reprogramming factors, but at this preliminary stage the project remains unfocused.

The novelty of this application is the development of cell lines from two human leukodystrophies, and the reprogramming of human oligodendrocytes into iPS cells. However, the applicant appears somewhat uninformed about myelin diseases in general. For example, s/he states that Canavan’s disease is a dysmyelinating disorder, which is not correct. The primary pathology is the vacuolation of white matter. S/he also cites periventricular leukomalacia (PVL) and cerebral palsy (CP) as glial-based diseases. A reviewer commented that this is not generally accepted, especially in the case of CP – there is white matter involvement, but the pathology is much more complex and heterogeneous. It is not entirely clear why the applicant chose the two diseases cited to generate iPS cells, nor what s/he will do with them if s/he succeeds.

The second aim of this project was the most criticized by reviewers. In this aim, the applicant proposes to derive iPS cells from oligodendrocytes. Reviewers commented that there is no evidence that oligodendrocyte-derived iPS cells would be any better for studying myelin disease than fibroblast-derived iPS cells from the same patient. This argument presupposes some sort of cellular memory that ensures preferred differentiation back to their previous state. While there is some suggestion that this might be the case, it is a very important research question and not something that can be assumed. In addition, the use of small molecules to induce oligodendrocyte dedifferentiation is very challenging, and the applicant presented no preliminary data to suggest its feasibility. Overall, therefore, reviewers disputed the feasibility of some parts of this aim, and they didn’t feel that cells generated from oligodendrocytes (the novel aspect of the work) will necessarily be any better than cells from other tissues.

For the first aim, the applicant presents good preliminary data on differentiation of cells into oligodendrocytes, and although s/he has not yet generated iPS cells, the review panel was not too concerned about feasibility. The final part of the grant uses straightforward techniques – transplantation into mouse models to analyze oligodendrocyte biology – and most reviewers weren’t concerned with feasibility. However, one reviewer commented that the experiments are not considered in sufficient detail. For example, intravenous delivery requires that the question of whether the cells enter the central nervous system at all be addressed, and the use of MRI to track migrating cells requires validation and proof. This same reviewer wondered what marker would be used in these experiments, as it was not discussed. Other reviewers commented that although the applicant is not an expert, the team described has the experience to do these experiments.

The applicant is a newly-appointed Assistant Professor at a top research institution, and is collaborating with two well-regarded co-investigators and several Research Scientists. This proposal is responsive to the RFA, as the proposed research should generate pluripotent human stem cell lines. The cells will be made available to others, as required.

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