Molecular mechanisms governing hESC and iPS cell self-renewal and pluripotency
A major problem in regenerative medicine today is that stem cells have the ability to cause tumors and in most cases we currently lack methods to make them safe. For example, two of the most promising stem cells for regenerative medicine, human embryonic stem cells (hESC) and induced pluripotent stem cells (iPS), both readily cause tumors in mice and there is every reason to believe they will do so in humans. 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. However surprisingly there is inadequate research into this fundamental problem and it is not funded to a significant degree by the NIH presenting a major gap in the field. In the proposed research we will address this problem by studying why hESC and iPS cells cause tumors and searching for new stem regulators that are safer. Our overall goal is produce safe hESC and iPS cell regenerative medicine therapies. One likely key culprit in the tumor forming capacity of these stem cells is a gene called Myc. Myc is a unique factor in the universe of 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. Myc has also recently been found to be a critical factor driving iPS cells to form tumors. However, we cannot simply eliminate Myc since it is important for efficient generation of iPS cells and likely for the maintenance of the positive properties of stem cells, including hESC, needed for regenerative medicine. In order to achieve our goal to enhance the safety of stem cells without sacrificing our ability to efficiently generate them or their key functions, we will take two main approaches in the proposed research. The first is to study how Myc works in iPS cells and hESC in order to find methods to enlist the positive effects while eliminating the negative properties. Remarkably, there is currently no information on how Myc functions in iPS cells and hESC. The second is to screen in a global, unbiased manner for new stem cell factors that can substitute for Myc or enhance iPS function independent of Myc. When these studies are successfully completed we will for the first time know the factors responsible for inducing stem cells to cause cancer, paving the way for eliminating that function, and we will have discovered new stem cell regulators that have better profiles of safety and efficacy than existing factors such as Myc. Together these achievements will bring us much closer to using the vast potential of iPS and hESC for new therapies that are both safe and effective. Longer term our goal is to work with our neural (Alzheimer’s disease, Parkinson’s disease, and spinal cord injury), cardiac, and liver disease teams here to generate safe and effective stem cell based therapies tailored for each patient.
Enhancing the safety of regenerative medicine therapies will be of great benefit to the State of California both in terms of improving the lives of patients, by removing arguably the most serious roadblock to regenerative medicine, as well as enhancing the knowledge of the stem cell field. It will also further the development and clinical use of regenerative medicine leading to a new, valuable biotechnology. California should be a leader in developing safe, effective regenerative medicine.
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.
During the current funding period we have made substantial progress toward achieving the goals of the three specific aims of the proposal. Toward the first aim, Test the hypothesis that Myc regulates hESC self-renewal and pluripotency, we have achieved several milestones. We have created hESC with Myc loss-of-function and determined a key role for endogenous Myc in regulating hESC self-renewal and pluripotency. We have also made substantial progress in addressing the objectives of the second aim, Study Myc regulation of hESC and iPS cell epigenetics. We have successfully completed the first phase of functional genomics studies (ChIP-chip) for endogenous c-Myc and N-Myc in hESC, finding a surprising dual role for Myc proteins in regulating the epigenetic state of hESC. In specific aim three, Discovery of novel enhancers and suppressors of human iPS formation, we have achieved two key goals already. First, we have created the novel cDNA library needed for screens and second we have begun conducting screens for pluripotency modulators. We have also found key new putative pluripotency factors in a novel proteomics screen. Together these studies have resulted in several key publications and demonstrate substantial progress after only two years of funding.
During the past year, our research has made substantial progress. In our efforts to better understand the function of the Myc proto-oncogene in human ES cells, we have identified a key novel cofactor for Myc in stem cells called Miz-1. Myc and Miz-1 have coordinate genomic functions such that while each factor can regulate gene expression independently, they frequently work in tandem together. When Myc and Miz-1 cooperate, their function together is most often to repress expression of differentiation-associated genes, particularly Hox genes. These data support a new model in which Myc maintains pluripotency in human ES cells and induces pluripotency in iPS cells by working with Miz-1 to keep differentiation genes turned off. Myc levels normally decrease during differentiation, which we theorize initiates a trigger allowing differentiation to proceed normally. High levels of Myc as are observed in cancer may permanently keep differentiation genes off contributing to the formation of the cancer. We have also found in our studies that Myc and Miz-1 genomic binding is associated with specific epigenetic states. When acting separately, Myc and Miz-1 bind to and maintain epigenetically active regions of the genome, but interestingly when they bind the genome together, they maintain a repressed epigenetic state. In other studies we have identified a novel factor that can substitute for Myc in iPS cell formation and we have made substantial progress in characterizing its function. In addition, we have identified novel protein cofactors of Myc that are specific to ESC that shed significant new light into how Myc functions to maintain pluripotency.
In the past year supported by CIRM funding we have made substantial progress in a number of areas related to stem cells and regenerative medicine. We published 6 papers including 5 supported by CIRM during this time.
We published only the second study ever on the metabolomics of induced pluripotent stem cells (iPSC), finding they are almost, but not entirely reprogrammed at the metabolic level to resemble embryonic stem cells (ESCs). Some notable differences between iPSC and ESCs include how they metabolized sugars. This study could have important implications for development of new iPSC production methods and improved iPSC safety and clinical efficacy.
We also published a study comparing the transcriptomes of iPSC and cancer cells. The transcriptomes are the total pattern of transcribed RNAs in cells, which can tell important stories about how the cells are programmed and functioning. Importantly, iPSC and cancer cells shared striking similarities in a number of ways in our findings including inhibition of differentiation and induction of specific metabolic programs. It was notable, however, that iPSC and cancer cells also differed in some important ways including high expression of pluripotency-related factors in iPSC was absent in the cancer cells. We also found that we could convert the cancer cells to behave more like iPSC through a form of cancer reprogramming that could have substantial import for developing new cancer treatments.
In another published study we reported the transcriptome that is regulated by Myc and its cofactor GCN5 in neural stem cells. Interestingly, Myc and GCN5-regulated genes were significantly overlapping, supporting the notion that these two factors often work together to regulate neural stem cell fate. However, the two factors also diverged in some ways suggesting that they work independently as well. Importantly, knockout of Myc or of GCN5 independently specifically in neural stem cells generated very similar phenotypes in mice. In each case the mice had very small brains, supporting a model in which Myc and GCN5 cooperate in neural stem cells to drive brain growth.
In the past year, the efforts funded by this award have yielded substantial progress in a number of areas. Our laboratory has published eight papers including a number of novel findings. We have made progress toward understanding how the Myc protein functions in stem cells and in cancer cells. In part this work has related to an important, yet poorly understood Myc co-factor called Miz-1. Our lab has determined how Miz-1 binds DNA and discovered two novel DNA motif sequences bound by Miz-1. Through these motifs Miz-1 strongly activates transcription of target genes. We are now trying to understand how this motif may related to Myc. We are also investigating how Myc may influence the global DNA methylation state of human embryonic stem cells. We have also determined that other factors in some ways act like Myc in that they are both oncogenes when overexpressed and factors that at normal levels play important roles in human pluripotent stem cells. More specifically we have found that the important pluripotency factors DPPA2 and DPPA4 are also, when expressed, able to cause cancer formation. This work published this year makes a big step forward in our understanding of the key relationship between stem cells and cancer cells. We have also investigated a novel factor called histone variant H3.3 and published two papers on this factor this year. Histone H3.3 is a fascinating molecule because it again is linked to both normal stem cell function and cancer. It is mutated in a devastating childhood tumor called glioblastoma. At the same time, our data suggests key normal roles for H3.3 in specific stem cells including germ stem cells that give rise to eggs and sperm. We published the first ever mouse knockout study of H3.3, which provided a window both into the normal and cancer-related functions of H3.3. We've also been studying how human ES cells and iPS cells behave in vivo in the brain with some fascinating results. Our overall goals are to enhance knowledge of stem cells and cancer and in so doing make stem cell-based regenerative medicine therapies safer and to catalyze the development of new cancer therapies. In the past year we have made a major leap forward toward these goals.
During the reporting period for this award our team has made substantial progress on the goals for this grant as reflected in four new publications. In a broad sense these studies have enhanced the field's understanding of the relationship between the molecular machinery in cancer and in stem cells. For example, key molecules that regulate normal pluripotent stem cell behaviors such as self-renewal and pluripotency also have roles in tumorigenesis and our studies have clarified how these regulatory factors (including chromatin and epigenetic factors Myc and histone H3.3) function both normally and in cancer. These studies will have impact both in the cancer stem cell field and for making safer stem cell-based regenerative medicine therapies.
More specifically in Aim 1 we have made progress in determining how H3.3 and Myc function in development and act to control gene expression through genomics studies. In Aim 2, we have made progress in understanding how Myc and its cofactors bind to the genome to regulate epigenetic states and gene expression in human pluripotent stem cells. In Aim 3 we have defined how key novel pluripotency-related oncogenic factors function including DPPA4 and DPPA2 via cutting edge genomics and proteomics studies.
- Cancer Cell (2013) Histone h3.3 mutations: a variant path to cancer. (PubMed: 24229707)
- Nat Med (2013) Scientists: you really need to get out of the lab more. (PubMed: 24013741)
- Methods Mol Biol (2013) Chromatin immunoprecipitation assays for Myc and N-Myc. (PubMed: 24006062)
- Regen Med (2013) Call for fellowship programs in stem cell-based regenerative and cellular medicine: new stem cell training is essential for physicians. (PubMed: 23477401)
- Biochem Biophys Res Commun (2013) Myc binds the pluripotency factor Utf1 through the basic-helix-loop-helix leucine zipper domain. (PubMed: 23665319)
- Epigenetics Chromatin (2013) Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development. (PubMed: 23570311)
- Stem Cells (2013) Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes. (PubMed: 23963736)
- Regen Med (2012) Key anticipated regulatory issues for clinical use of human induced pluripotent stem cells. (PubMed: 22830621)
- Stem Cells Dev (2012) Induced pluripotency and oncogenic transformation are related processes. (PubMed: 22998387)
- PLoS One (2012) Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism. (PubMed: 23077522)
- Cell Stem Cell (2012) Utf1: Goldilocks for ESC Bivalency. (PubMed: 23217417)
- PLoS One (2012) N-Myc and GCN5 regulate significantly overlapping transcriptional programs in neural stem cells. (PubMed: 22745758)
- J Vis Exp (2011) Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety. (PubMed: 21808229)
- Epigenetics Chromatin (2011) Myc and Miz-1 have coordinate genomic functions including targeting Hox genes in human embryonic stem cells. (PubMed: 22053792)
- Cell Stem Cell (2011) Inducing iPSCs to escape the dish. (PubMed: 21816362)
- Differentiation (2010) myc maintains embryonic stem cell pluripotency and self-renewal. (PubMed: 20537458)
- Cancer Res (2009) Acting locally and globally: Myc's ever-expanding roles on chromatin. (PubMed: 19773445)
- Stem Cells (2009) Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine. (PubMed: 19415771)
- PLoS One (2009) N-Myc regulates expression of pluripotency genes in neuroblastoma including lif, klf2, klf4, and lin28b. (PubMed: 19495417)
- Nature (2009) Journal club. A cell biologist looks at the risk and promise of a new insight into stem cells and cancer. (PubMed: 19158746)