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RL1-00655-1: Large-Scale Reprogramming of Human Somatic Cells to a Pluripotent Stem Cell-Like State

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Pluripotent stem cells can by definition give rise to all cells of an organism and also self-renew. These properties have long been evident in embryonic stem cells isolated from early stage embryos of many species, and then maintained in culture with the aid of exogenous factors that promote proliferation in the absence of differentiation. Advances in isolating and propagating human embryonic stem cell lines have spurred enthusiasm for applying these cells to studies of disease mechanisms, to screening of more effective drugs, and to development of new therapies for disease or and injury. However, progress has been hindered by continuing ethical debates over the theoretical fates of such embryos. Therefore, there is high interest in alternative technology to reprogram differentiated somatic cells to a pluripotent-like state that has the potential to ultimately circumvent dependence on embryos. This has already been demonstrated to varying degrees of efficacy, but each of the current strategies has significant limitations. The objective of this proposal is to establish robust, efficient and high-throughput processes for reprogramming donor somatic cells into pluripotent stem-like cells. An important component of this effort will be to extend these processes through cell derivation and maintenance in a way that could expedite the banking of pluripotent cell lines that may have future clinical utility. Our approaches are both pragmatic and novel. Thus, we will focus on optimizing, comparing and then extending the capabilities of several established reprogramming strategies. Our goals are to (1) test the hypothesis that providing a qualitatively and quantitatively more potent reprogramming stimulus can improve reprogramming efficiency; (2) test the hypothesis that optimizing the source of primary human somatic cells can improve reprogramming efficiency; (3) test the hypothesis that the efficiency of derivation and recovery of pluripotent cell lines can be improved by providing a more permissive and supportive environment for cells during the nuclear remodeling and reprogramming process; ( 4) lay the initial foundation for establishing a large homozygous pluripotent stem cell bank that can meet most potential clinical needs by incorporating the optimized methods into a cGMP-compliant process at the earliest opportunity.

Statement of Benefit to California (provided by applicant)

Severe injuries and degenerative diseases are leading contributors to healthcare-related costs that affect all Californians. The potential to derive new types of therapeutics from pluripotent stem cells could revolutionize healthcare by providing more effective and perhaps curative treatments. California is a major hub for research and development on stem cells. In recent years, thought-leaders in this field from around the country have relocated to California universities and biotech companies. Major investments in early-stage companies by venture capital firms and pharmaceutical companies have been announced to rapidly exploit and develop discoveries made by academic researchers. If CIRM-funded research continues to spur progress, this area of biotechnology has the potential to grow substantially. This will help to fulfill CIRM’s goals of increasing the availability of new stem cell-based therapies and diagnostics to citizens, create new jobs and keep the state at the forefront of the biotech industry.

Review

The principal investigator (PI) for this application proposes to establish a robust, efficient and high-throughput process for reprogramming somatic cells into induced pluripotent stem (iPS) cells. First, the PI proposes to test several reprogramming strategies that involve the fusion of fibroblasts with extracts prepared from normal or genetically manipulated pluripotent cells. Second, the applicant proposes that some cell types may be easier to reprogram than others and intends to examine sources other than fibroblasts to optimize iPS cell generation. Additionally, the applicant proposes that current culture conditions for the derivation of iPS cell lines limit the efficiency of the process, thus the team will use a culture system they have developed for human embryonic stem cells (hESC) to improve iPS cell generation. Finally, the proposal is to move these optimized iPS cell generation methods into a process that is compliant with current good manufacturing practices (cGMP), with the goal of generating autologous iPS cell lines or establishing a HLA-homozygous pluripotent stem cell bank for clinical needs.

Reviewers were in agreement that improving somatic cell reprogramming is an important goal. Reviewers also agreed that the most novel aspect of the proposal is the use of a strategy that avoids the presence of viral vectors and transgenic DNA in reprogrammed cells. However, reviewers expressed serious concern that this strategy is very risky, and the applicant does not provide preliminary data supporting the feasibility of the proposed work. The PI should have taken into consideration the failed attempts by others and tried to address them. Furthermore, there is no supporting rationale for the use of cell types other than fibroblasts for the reprogramming protocol. For one of the aims, the PI proposes to derive pluripotent cells from the rare population of cell donors that are homozygous for HLA types. A reviewer criticized that the applicant did not identify the source of such cells. There is no letter of support from any other group or organization supporting this endeavor. Finally, reviewers discussed the lack of information on how the proposed processes will be made cGMP-compliant, and the lack of the PI’s expertise with hESC and iPS cells. Taking into account these shortcomings, the overall enthusiasm for this proposal was low.

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