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RL1-00637-1: Induced Pluripotent Stem Cell Lines from Retinal Pigment Epithelial Cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Human embryonic stem cells (hESC) have great potential for the treatment of disease and injury because they are pluripotent in their capability to form most cell types in the body. They will also be of great utility for screening new drug candidates, and for understanding the molecular mechanisms of human development and disease. However, a big challenge in the field of stem cell research is to develop patient-specific, immunologically matched hESC. Recently, it has been shown that both fetal and adult human somatic cells can be reprogrammed to induce a pluripotent state via expression of specific sets of transcription factors. Thus, a future therapeutic option would be to obtain a patient’s own skin cell, convert it to a cell resembling an embryonic stem cell, called an induced pluripotent stem (iPS) cell, and then use that cell for therapy. However, the iPS cells generated thus far are unsuitable for therapies because retroviral DNAs that integrate into the genome were used, which may cause mutations by inserting into a gene and altering it in a negative way (some cancers are caused by this mechanism). There is a critical need in the field to generate iPS cells using non-integrative reprogramming methods. We have obtained preliminary data suggesting that retinal pigment epithelial (RPE) cells can be reprogrammed to resemble hESC using a method that avoids the danger of integration of DNA into the genome. This application proposes to further develop this new technology to allow generation of iPS cells from RPE, which are easily obtained from biopsies. A second goal is to obtain iPS cells that differentiate into eye cells treatment of eye disease. The new cell lines we generate will be carefully characterized with respect to their capabilities for forming different useful cell types. They will be compared to hESC, which remain the gold standard for pluripotent stem cells, and other iPS cells. The propensity for differentiation into RPE and other ocular cells will be explored. If successful, these experiments will provide a method to obtain patient-specific cells that might be useful in therapies for a variety of maladies, including eye diseases such as age-related macular degeneration.

Statement of Benefit to California (provided by applicant)

The State of California, like the rest of the nation, faces immense challenges to its health care system, with soaring medical costs due in part to continuing care of our aging population. The percentage of elderly in California is expected to grow from what was 14 percent in 1990 to 22 percent in 2030. Chronic degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, age-related macular degeneration, cancer, diabetes, cardiovascular disease, osteoarthritis, and osteoporosis afflict a growing number of individuals in California. Major innovative approaches are now, more than ever, an imperative. Human embryonic stem cells (hESC) have great potential for the treatment of disease and injury because they are pluripotent in their capability to form most cell types in the body. They will also be of great utility for screening new drug candidates, and for understanding the molecular mechanisms of human development and disease. However, a big challenge in the field of stem cell research is to develop patient-specific, immunologically matched hESC. Recently, it has been shown that both fetal and adult human somatic cells can be reprogrammed to induce a pluripotent state via expression of specific sets of transcription factors. Our proposed research will develop new methods for generation of pluripotent stem cells from adult somatic cells, and identify new cell types for the treatment of eye diseases such as age-related macular degeneration. If successful, this work will be a great benefit to the state by providing useful new technology that addresses a critical need in the field of stem cell research. In addition, it provide new approaches for therapies to treat degenerative conditions that afflict millions of Californians.

Review

The goal of this proposal is to develop a novel gene transfection technology that avoids the use of viruses for generation of induced pluripotent stem (iPS) cells. The proposed approach employs a novel, particle-based transient transfection system for the delivery of reprogramming genes. The method will be developed with retinal pigmented epithelial (RPE) cells, yielding a valuable tool for the study and treatment of macular degeneration. Experiments are proposed to refine and optimize methods for non-integrative reprogramming of adult RPE cells which are readily obtained from biopsies.

This proposal is highly significant, because if successful, it could eliminate the need for retroviral vectors and their associated potential for mutagenesis. The use of the proposed technology for delivery of reprogramming genes is innovative, and the value of this approach will be heightened if the method is shown to be generally applicable to a variety of starting adult cell types.

Preliminary data suggesting the generation of iPS cells from human embryonic fibroblasts via the novel transfection method appear promising. However, reviewers viewed these results as extremely preliminary and had strong reservations about the use of proposed particle transfection system. Aspects of serious concern included the non-degradable nature of particles and possible secondary effects from their persistence in cells, the lack of information about gene dosage or degree of DNA conjugation with the particles, and questions about the efficiency and reproducibility of the novel transfection system.

The proposal describes a well designed and rigorous methodology to evaluate the pluripotency of derived cells. Reviewers were concerned about proposed alternative strategies which were viewed as too vague and not convincingly feasible.

The PI and named co-investigators have appropriate expertise and experience to perform the proposed experiments. The planned collaboration with a biotech company strengthened the proposal. The low percent effort committed by a co-investigator who is a leading stem cell expert raised reviewers’ concerns as to whether he/she would be significantly involved in the project.

The proposal is responsive to the RFA. It could provide a valuable approach for generation of cell lines and yield clinically useful new lines. The full identities and potential of derived cells and cell lines that might be produced by this technique remain to be determined, and the lack of sufficient preliminary analysis decreased reviewer enthusiasm for the proposal. The plan to deposit and share new cell lines appeared adequate.

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:

• Duncan, Ian
• Zwaka, Thomas