Genetic dissection of human cellular reprogramming to pluripotency
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.
Specialized cells can be reverted back to induced pluripotent stem cells (iPSCs), and this “reprogramming” holds enormous promise for drug discovery as well as cell transplantation approaches towards the treatment of degenerative diseases. Our goal is to understand the genes and pathways that regulate the process of generation of human iPSCs. During the first year of this award we have made a number of advancements on this front. We have used a large-scale genetic approach to discover new genes and cellular pathways that operate in the specialized cells and act to oppose the process of reprogramming. We have used sophisticated computational biology approaches to gain further insights into these pathways. We have also began the characterization of novel reprogramming barriers at the molecular and cellular level. For example, we have discovered that proteins operating outside of the cell and at the cell surface have unexpected functions in the process of generation of iPSCs. These results keep us on track towards the goal of achieving a deeper understanding of the process of reprogramming human cells to the pluripotent stem cell state.
Specialized cells can be reverted back to induced pluripotent stem cells (iPSCs), and this “reprogramming” holds enormous promise for drug discovery as well as cell transplantation approaches towards the treatment of degenerative diseases. Our goal is to understand the genes and pathways that regulate the process of generation of human iPSCs. During the second year of this award we have made major progress toward this goal. We have discovered several novel cellular pathways that operate in specialized cells and act to oppose the process of reprogramming. We have characterized some of these key pathways in detail, including their timing of action, how they affect the reprogramming process, and how they interact with one another. These results keep us fully on track towards the goal of achieving a deeper understanding of the process of reprogramming human cells to the pluripotent stem cell state.
Human iPS cells provide a new platform to investigate disease etiology and devise novel therapies in Regenerative Medicine. The objective of our proposed research is to gain a deep understanding of the molecular mechanisms that underlie reprogramming of human somatic cells to the iPS cell state. Our proposal is based on a genome-wide functional screen in the process of human iPS cell generation that was recently carried out in our lab. This has been a rich source of data that we have mined in several different and very insightful ways. The specific goals of this research were to identify novel pathways that act as barriers to human iPS cell generation, and to dissect the mechanism of action of one of those barriers. Both goals were achieved and surpassed, and therefore this research can be considered successfully completed. The results uncover novel barriers to reprogramming and the complex ways by which they cooperate to oppose dedifferentiation, with important implications both for Regenerative Medicine and Cancer Research. Our findings also shed light on alternative states of human pluripotency.
Our progress to date includes two publications reporting part of our findings:
1. Qin et al., “Systematic identification of barriers to human iPSC generation.” Cell 158:449 (2014).
2. Diaz et al., “HiTSelect: a comprehensive tool for high-complexity-pooled screen analysis.” Nucleic Acid Research 43(3):e16 (2015)
A 3rd publication arising from this research is currently under review.
- Cell Rep (2016) YAP Induces Human Naive Pluripotency. (PubMed: 26947063)
- Nucleic Acids Res (2015) HiTSelect: a comprehensive tool for high-complexity-pooled screen analysis. (PubMed: 25428347)
- Cell (2014) Systematic Identification of Barriers to Human iPSC Generation. (PubMed: 25036638)