Tools and Technologies II
Human embryonic stem cells (ESCs) have the unique property of being pluripotent: they have the capacity to develop, or ‘differentiate,’ into most of the body's cell types in the petri dish, which makes them an immensely powerful tool for studying how certain diseases arise and for finding drug candidates that might combat these diseases. They also hold the promise to one day enable cell replacement therapies, in which the function of tissues that have been lost or damaged due to disease or injury is restored by transplantation of stem cell-derived differentiated cells. A few years ago, researchers discovered how to derive such pluripotent stem cells from non-pluripotent cells, for example from a person's skin cell. The method of, in effect, de-differentiating an adult cell back to a pluripotent state is called reprogramming, and the resulting cells are so-called induced pluripotent stem cells, or iPSCs. By obtaining iPSCs from patients, researchers have already begun to study the molecular mechanisms responsible for the development of some diseases. iPSCs are generated by forcing non-pluripotent cells to produce several proteins known as reprogramming or pluripotency factors, usually by infecting the cells with viruses that have been engineered to integrate the DNA encoding these factors into the genome of the cells. As one might imagine, this process can lead to mutations. What is more, these factors must eventually be turned off completely for the pluripotent cell to differentiate, a process that is not always efficient. When the factors remain turned on, the cells can become cancerous, and grafting virus-derived iPSCs carries certain risks. Our research aims to develop a new tool for virus-free derivation of iPSCs, namely by introducing into cells artificial messenger RNAs encoding the pluripotency factors . These RNAs do not incorporate into the cells’ genomes and are eventually degraded by the cells once reprogramming is complete. If we succeed, researchers and clinicians can more safely reprogram cells, bringing iPSC-based cell replacement therapies a major step closer to implementation. Differentiating ESCs or iPSCs into particular cell types in the petri dish – say, into a so-called dopaminergic neuron, the type of neuron that is lost in patients with Parkinson’s disease – requires the carefully timed and dosed application of growth factors and the manual isolation of cells with the desired properties. Often even our best protocols yield cell populations with only a small fraction of the desired cells. In what is another application of our RNA-based approach, we have developed RNA molecules that can sense the presence of so-called microRNAs, whose presence marks particular cell types, and we aim to show that these RNA sensors can be used to sort out the desired cells from the unwanted ones. Thus, both of our goals promise to help bring stem cells a significant step closer to their clinical use in patients.
Statement of Benefit to California:
Our proposed research ultimately aims to establish new procedures for the safer and more efficient derivation of stem cell-based cell preparations for use in cell replacement therapies. The California Institute for Regenerative Medicine has recognized the urgent need for technologies that accelerate the translation of basic stem cell research into clinical application, and our proposed research is addressing this need. The technology we seek to launch will benefit California in several ways. Our methods represent an enabling technology that has the potential to accelerate both basic and translational stem cell research conducted in California, thus reinforcing and expanding California’s eminence in the field. Since its utility is not limited to a particular disease area, our technology holds the promise to find widespread application in stem cell laboratories across the state. In addition, the tools, methods and reagents we seek to develop will lower the barrier of entry into stem cell research for scientists from neighboring areas of expertise, thereby leveraging California’s existing stem cell infrastructure. More importantly, however, by promising to make the derivation of patient-specific induced pluripotent stem cells more efficient, our proposed research promises to speed up the development of stem cell-based disease models, thus facilitating the establishment of drug screens and accelerating drug development. Our innovative non-genetic method to purify desired types of cells from mixed populations without the need to raise cell-type specific antibodies has the potential to become widely used as a routine quality control step in the derivation of purified cell preparations for grafting. Given that a growing number of Californians are afflicted by degenerative diseases for which cell replacement therapy might be of therapeutic benefit, our proposed research may have a direct positive impact on the quality of life of millions of Californians.
This proposal is focused on the development of novel tools for virus-free generation of induced pluripotent stem cells (iPSCs) and live cell monitoring of neuronal subtypes differentiated from pluripotent cells. The applicant proposes to use RNA-based technologies to address two bottlenecks to stem cell translation: the lack of highly efficient, integration-free methods for iPSC generation and the lack of tools for the identification and segregation of differentiated live cells of specific lineages from mixed cell populations. Three specific aims are proposed: (1) to develop methods for RNA-directed protein expression in human cells; (2) to reprogram primary human fibroblasts into iPSCs via RNA delivery; and (3) to develop and validate microRNA (miRNA)-based sensors for the visualization and segregation of specific neuronal subtypes differentiated from pluripotent cells. Reviewers appreciated the significance of the translational bottlenecks addressed but found this proposal to be only minimally innovative. Following submission of the application, another group published success with RNA-based iPSC reprogramming, effectively accomplishing the majority of the first two Aims of this proposal. While reviewers acknowledged that there are novel aspects to the applicant’s approach and a need for further work on RNA reprogramming, the recent publication and its subsequent implementation by several other groups significantly reduced the novelty of this proposal and dampened the reviewers’ enthusiasm. Additionally, while Aim 3 was generally well received, reviewers noted that the concept of miRNA sensors is only novel in its proposed application to human pluripotent cells and their derivatives and were uncertain that the tool developed in this aim could be used for translational work. The reviewers found the research plan to be carefully designed to give meaningful results while noting that the completion of Aims 2 and 3 depends on the success of Aim 1, in which RNA delivery will be optimized. Reviewers found the preliminary data exciting, but were concerned by the absence of key data showing that RNA transfection would not evoke an interferon response and cellular toxicity. They appreciated the detailed description of methods and the discussion of pitfalls and alternative approaches. The reviewers found Aim 3 the most interesting -- to develop miRNA sensors of specific neuronal subtypes -- but did not support funding of this aim on its own given its dependence on work proposed in Aim 1, the limited preliminary data supporting this aim, and uncertainty that it meets the objectives of the RFA. Following submission of this proposal, the Principal Investigator (PI) requested a change of PI to another member of the proposed team. The original PI indicated a continued commitment to the project in a supporting role with a reduced percent effort. The reviewers would have preferred to have the original PI lead this project, given his/her exceptional track record and stature in the field. However, they noted that the new PI has had excellent training and has published several papers almost wholly concerned with RNA, RNA processing or miRNAs. In general, reviewers found that the change of PI detracted from their overall level of enthusiasm for the project, but considered this a relatively minor issue and described the research team as well suited to perform the proposed experiments. Overall, while reviewers appreciated the significance of the bottlenecks addressed in this proposal and the carefully designed research plan, they raised significant concerns about the novelty of the approach and potential impact of the proposed research. Because of these concerns, the reviewers did not recommend this application for funding.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.
- Stephen L Minger