The reprogramming of skin fibroblasts into pluripotent stem (iPS) cells represents a significant milestone towards the goal of developing stem cell therapies tailored to the patient. However, the reprogramming is slow and inefficient. A key step is the ability of four exogenously introduced transcription factors to turn on or activate the endogenous versions of their own genes, a process termed autoregulation. In fibroblasts, the genes for the endogenous transcription factor genes are turned off because they are assembled into a repressive structure termed silent chromatin. The repressive structure must be removed to activate the endogenous stem cell transcription factor genes. The efficiency by which the endogenous genes are activated is poorly understood. Dozens of different proteins assist the four transcription factors during the reprogramming process. However, little is known of how the dozens of proteins coordinate their actions to remove the silent chromatin structures and replace them with active chromatin. Our goal is to study this process and use the resulting knowledge to improve the efficiency of iPS cell formation.
This process can be studied by recreating it in a test tube or in vitro. This biochemical recreation involves three steps: 1. Generating highly pure versions of the four transcription factors. 2. Reproducing the silent chromatin environment of the gene. 3. And finally, identifying each of the steps involved in reactivating the endogenous genes. We have developed a technology that permits us to biochemically recreate silent chromatin on the transcription factor genes in vitro. We then attach the silent chromatin to magnetic beads, add protein mixtures from disrupted stem cells, and use magnets to capture beads with the attached proteins, which are in the process of converting silent chromatin to active chromatin. This “immobilized template capture” technique also allows us to delineate the steps involved in the reprogramming process. The proteins are identified by a state-of-the-art method known as multidimensional protein identification technology (MuDPIT). By combining the immobilized template and MuDPIT techniques, we will be able to provide detailed knowledge about how autoregulation is achieved. As such, we will be able to determine which specific steps are limiting in the conversion of fibroblast to iPS cells. Knowledge of the mechanism of stem cell gene regulation is essential for fully understanding stem cell self-renewal and the transition of fibroblasts to iPS cells. The information can be utilized to improve the efficiency of iPS cell formation.
Statement of Benefit to California:
California is investing 6 billion dollars in stem cell research with much of the principal being spent in the first 10-15 years on research and development. To date, much research has focused on the therapeutic potential of stem cells and understanding a few fundamental aspects of how stem cells self renew and differentiate. A major gap in our understanding lies in the most basic aspect of stem cell biology, the mechanics by which stem cell genes are regulated. To fully understand and to optimally manipulate the technology, a significant amount of basic knowledge must be obtained.
The lack of basic mechanistic knowledge is what initially limited gene therapy and immunotherapy from achieving clinical application. For example, in gene therapy, we knew how to design therapeutic genes but knew little about how to deliver and regulate them. The situation has improved considerably in recent years, driven not by new technologies per se, but due to an investment in understanding basic mechanisms. We are now starting the same process with stem cells. Our technology and knowledge base have improved considerably but there are large gaps. Few scientists doubt the potential. However, many overestimate our knowledge base and ability to achieve therapeutic goals. It is important that we determine at the most basic level how stem cell genes are regulated.
We know that four gene regulatory factors, when added to skin fibroblasts, can convert these into stem cells. However, we do not know precisely how they do this. To date, CIRM has invested heavily in labs trying to understand the functions of these four transcription factors in living cells. However, in the gene regulation field of biology, many of the major advances derived from biochemical or in vitro studies.
My laboratory works on the fundamental aspects of how genes are regulated. We have recreated key events in a test tube. We have developed new biochemical approaches for determining how stem cell genes are regulated and for understanding how the four transcription factors function when bound to chromatin. Our studies will provide insight into and understanding of the processes by which stem cells self renew and differentiate. As such, the basic knowledge will help further the transition of into stem cells into clinical applications.