Year 1

The key hurdle in moving regenerative medicine to the clinic is that stem cells have the ability to cause tumors and in most cases we currently lack methods to make them safe. Two of the most promising stem cells for regenerative medicine, human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPS), both are almost certain to cause cancer in humans if transplanted. The reality is that if we cannot prove that stem cells are safe and do not cause tumors, they will never be used in patients. In the first year of our research we have made substantial progress to address this problem by studying why hESC and iPS cells cause tumors, focusing on the role of the proto-oncogene Myc, and searching for new stem regulators that are safe. During this time we have had four publications and another one is in review.

Myc is unique amongst stem cell regulators because it not only has key roles in the normal, positive functions of many stem cells, but also when found in excess it is one of the most potent cancer-causing genes in humans. During the first year of research we have made major headway in understanding how Myc functions in regulating the biology of hESC, the objective of Aim 1 of the award. To this end we have taken two approaches, studying Myc function in hESC and also in mouse ESC (mESC). The mESC are a powerful, complimentary tool to the hESC. In both hESC and mESC we have found that Myc genes are essential for normal pluripotency and self-renewal. Myc genes encode proteins that are transcription factors that control hESC behavior through regulating levels of other factors. We have found that Myc directs ESC metabolism and proliferation as well as potently suppresses their differentiation, together in this way maintaining their stem cell state. In the course of these studies we have identified specific molecules and pathways downstream of Myc that represent candidate mechanisms by which it orchestrates pluripotency and self-renewal. Interestingly, some but not all of these factors act downstream of Myc in tumors as well. Thus, one theory is these molecules may hold the key to teasing apart tumorigenicity from pluripotency.

In its job as a transcription factor, Myc binds to DNA in stem cells and regulates the structure and function of the DNA through a process called epigenetics. Very little is known about how Myc regulates epigenetics in ESC and iPS cells. In the first year of this award we have conducted a preliminary global study of where the two main Myc proteins, c-Myc and N-Myc, bind in the hESC genome and the epigenetic events associated with that binding using a powerful methodology called functional genomics. We have also conducted biochemical studies of these epigenetic events with intriguing preliminary data. In addition we are working to investigate Myc epigenetic function in human iPS cells.Together this is substantial progress toward the goals of Aim 2 of the award.

Our goal in Aim 3 is to discover new stem cell regulators, both factors that positively and negatively regulate self-renewal and pluripotency. Toward the specific objective of finding factors that suppress stem cell biology, we have conducted two preliminary human iPS cell based screens using a tool called an shRNA library, which is commercially available. While we are still optimizing this screening approach, the two initial experiments have given us extremely valuable information about the optimal way to conduct the screen in the future and have demonstrated in principle that the screening methodology works. Moving toward the specific goal of finding positive regulators of pluripotency, we found that the key tool we need, something called an hESC cDNA retroviral library, does not commercially exist. Thus, a crucial goal is to synthesize this tool ourselves and we are actively working foward on that. This library is likely to be an extremely valuable tool for other stem cell researchers as well. Finally, we have also been employing an exciting proteomics-based methodology to screen for factors that can substitute for Myc in regulating stem cells including both ESC and human iPS cells. The objective in this area is to find factors that can do the job of Myc in regulating stem cells but without its tumor promoting properties. Our proteomics-based studies in the first year of the award have yielded some very interesting preliminary candidates in ESC, including some known, important pluripotency regulators.

We are confident that in the second year of the award we will further build on the substantial progress we have made in the first year, gaining additional momentum toward our goal of safe and effective stem cell-based regenerative medicine therapies.