It is now possible to turn adult differentiated cells from a patient into human induced Pluripotent Stem (iPS) cells. These iPS cells hold enormous promise for new therapies in Regenerative Medicine, because they can be coaxed in the laboratory to become any cell type in the human body. While this is a spectacular property, we understand very little about the basic biology of generation of human iPS cells. This is a major gap in our understanding of arguably the most promising new avenue in Regenerative Medicine today. An understanding of the molecular and cellular processes that govern iPS cell generation will be essential for the development of
safe and efficient therapies using iPS cells in the clinic.
Research in biological model organisms like fruit flies or mice has shown that unbiased genetic screens, where the entire genome is assessed to discover genes that regulate particular processes, are an enormously powerful tool in basic biology studies. Gene silencing using RNA interference (RNAi) opens the opportunity to perform genetic screens in human cells. Our lab has recently carried out an unbiased genome-wide RNAi screen in the process of turning human skin cells into iPS cells. This extensive screen has already revealed several novel genetic regulators of human iPS cell generation, some of which have been preliminarily studied further. We propose to follow-up on this RNAi screen to gain a comprehensive understanding of the basic biology of human iPS cell generation.
Pluripotent stem cells may provide new treatments for devastating and presently incurable conditions such as diabetes, Parkinson's disease, muscular dystrophies, spinal cord injuries, and many other diseases. The ability to generate induced Pluripotent Stem (iPS) cells from differentiated cells of an adult patient provides a major new tool to study degenerative diseases in the lab, discover new drugs, or develop cells for transplantation. However, we lack an understanding of the basic biological mechanisms that underlie the generation of iPS cells. Our proposal aims to use an unbiased approach to understand the genes that regulate the generation of human iPS cells.
The development of human pluripotent stem cell-based therapies will significantly increase the options available in the California health care system. These new therapies are expected to reduce the long-term health care costs to California by providing cures to degenerative diseases that are currently chronic and require expensive periodic treatment.
Our research is also expected to stimulate the development of biotechnology industry focused on clinical applications of iPS cell technology. Such development will be of great benefit to California by attracting high-skill jobs and tax revenues, and by making the State a leader in a field that is poised to be the economic engine of the future. The State of California will also stand to benefit from the intellectual property generated by this research.
This proposal is based on a genome-wide screen, performed in the applicant’s laboratory, to identify genes that play a role in reprogramming human fibroblast cells to induced pluripotent stem (iPS) cells. The goal of Aim 1 is to validate many of the top hits, thereby identifying genes that act as enhancers or barriers to reprogramming. Aim 2 is focused on a particular gene, already validated as acting as a barrier to iPS cell formation, and seeks to investigate its specific role in cellular and molecular mechanisms during the reprogramming process.
Significance and Innovation
- The overall significance of the proposed work lies in its potential impact on understanding the reprogramming process; there may be practical outcomes such as the identification of chemicals that promote human iPS cell generation or the development of tools for sorting intermediate cell types for mechanistic studies.
- Innovative aspects of this work include the strategy that was used for the original screen, the analysis of genes that both positively and negatively affect reprogramming, and some of the specific stem cell assays employed.
- The hypothesis regarding the function of the gene pursued in Aim 2 is interesting and novel.
- In the view of some reviewers, but others disagreed, the applicant overemphasized the potential impact of the proposed work on translational and clinical uses of iPS cells. Many important basic questions remain to be answered in pluripotent stem cell biology, but they do not represent critical roadblocks toward applied research.
- While reprogramming efficiency, a focus in this application, is widely pursued in the field, iPS cell quality may in fact be more important and, although mentioned, deserves more experimental attention in this proposal.
- The gene picked for follow up studies in Aim 2 afforded a 4-fold increase in reprogramming efficiency when knocked down. This relatively small effect in the context of a genome-wide screen dampened the enthusiasm of some reviewers. Others appreciated that the screen was sensitized such that the applicant was able to pick up factors with small effects, perhaps better enabling discovery of inhibitors of reprogramming.
Feasibility and Research Design
- The screen is well described and has been completed. This and the comprehensive nature of the screen serve as a strong basis for the proposed experiments.
-The proposed approaches are logical and preliminary data support feasibility.
- There is some concern that only a fraction of the genes identified in the screen are truly involved in reprogramming. Elimination of false-positives must be rigorously pursued; this may not have been the case and was not discussed properly. In this context it is of note that many identified genes play roles in general cell homeostasis, and may thus not have functions specific to the reprogramming process.
Principal Investigator (PI) and Research Team
- The PI is a well-established, outstanding investigator with an excellent publication record.
-The PI has made several important contributions to our knowledge of reprogramming and has the expertise to conduct the proposed research.
- The team is very strong.
Responsiveness to the RFA
- The application is responsive to the RFA; it is focused on basic aspects of human stem cell biology.
- PROGRAMMATIC DISCUSSION
- A motion was made to move this application into Tier 1, Recommended for Funding. During a brief discussion it was reiterated that this proposal is based on an interesting screen and is likely to have a broad impact on stem cell biology. The motion carried.