Basic Biology I
Stem cell quality and safety in developing regenerative medicine therapies is of utmost importance. Poor outcomes include inadequate functionality, exhaustion, immune rejection, cancer development, and others. Recent studies strongly support our core hypothesis that mitochondrial function determines stem cell quality and safety. Dysfunctional mitochondria foster cancer, diabetes, obesity, neurodegeneration, immunodeficiency, and cardiomyopathy. Unlike whole genome approaches, methodological hurdles for evaluating mitochondria in human embryonic stem cells (hESCs) and in reprogrammed human induced pluripotent stem cells (hiPSCs) are significant and techniques developed or adapted for stem cells are almost non-existent. With a 2-year CIRM Seed Grant, we developed new approaches for analyzing respiration (oxygen consumption that drives energy production) in hESCs in a series of 4 invited publications for the stem cell scientific community (www.JoVE.com; 2008). A manuscript describing the function of hESC mitochondria in low oxygen (hypoxia), in normoxia (room air), and during differentiation is in final preparation. We also collaboratively developed small molecule inhibitors of specific mitochondrial functions, thereby providing new essential tools to the scientific community for interrogating the function of stem cell mitochondria. Unlike current inhibitors of mitochondrial function, with are generally non-specific, irreversible, and toxic over time, our novel inhibitors are reversible, non-lethal, and target a range of specific mitochondrial functions. These inhibitors are undergoing continuous molecular refinement and validation studies for use in basic studies and can potentially lead to insights for clinical application in common diseases, such as diabetes and cancer. These advances form the underpinnings for our current proposal. We now propose two main aims to address fundamental questions in mitochondrial biology and safety of stem cells. In Aim 1, we have identified two lead candidate mechanisms for regulating oxygen utilization and energy production in stem cell mitochondria. It is known that reduced levels of the proteins that regulate energy production favor the development of cancer. We will determine the functionality of energy producing pathways in hESC, hiPSC, normal cell, and cancer cell mitochondria. In Aim 2 studies, we will utilize our novel mitochondrial inhibitors to determine which additional functions are essential to derive safe stem cells for clinical development. We also embark on a focused discovery protocol, based on already established genome-wide methods for stem cells, to identify key regulators of mitochondrial maturation with stem cell differentiation. Combined, our studies build upon successful CIRM-funded work to move into functional analyses of mitochondria that support stem cell self-renewal, survival, and differentiation, with major economic and social implications for new-age cellular therapies in medicine.
Statement of Benefit to California:
Our proposal benefits the people and state of California by adding new essential knowledge on the mitochondrial functions of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and their lineage differentiated derivatives, in support of the California peoples' and taxpayers' commitment to personalized cell therapies. This new work builds on a highly successful two-year CIRM Seed Grant,[REDACTED], that provided one of the first systematic characterizations of stem cell mitochondria. CIRM funds supported 1) four published protocols (www. JoVE.com; 2008) on growing pure stem cells and methods of characterizing mitochondria for the scientific community, 2) the first report of high-resolution genome differences between hESCs derived from different individuals (Stem Cells; 2008), and 3) now almost complete studies that characterize the functional capabilities of hESC mitochondria that are being prepared for publication(s). This CIRM-supported comprehensive characterization of stem cell mitochondria function , and new mechanism- and discovery-driven studies in our current proposal, will help guide clinicians and scientists to select the best possible stem cells for investigation and use. Our ongoing work will propel therapy development in California’s major academic centers and will provide information to many of California's biotechnology and pharmaceutical companies in the ever growing stem cell industry, whose success will propel hiring and increased economic prosperity for the state. Results from these studies will provide additional information to patient advocates, ethicists, and medical geneticists to help select the optimal course for developing and modifying stem cell usage policies and infrastructure within California. This proposal will also provide new information for patients and their physicians that may, at some future time, impact the selection of particular stem cells with specific mitochondrial attributes for specific types of therapeutic applications. In sum, added knowledge provided by our proposed studies on mitochondrial factors that control stem cell metabolism and mitochondrial maturation will help define and drive successful methods of hESC and hiPSC differentiation, will identify the most completely reprogrammed hiPSCs from a metabolic standpoint, and will generate cell therapies with reduced risk, increased safety, and limited cancer potential. With success tangible health and economic impact on California, its academic institutions and biotechnology/pharmaceutical companies, and the rest of the nation will be achieved as California and its people lead the way forward with personalized medicine for the 21st century and beyond.
The goal of the proposed research is to understand the role of mitochondria and the cellular energetic state in stem cell fate decisions. The first specific aim will focus on identifying mechanisms that regulate changes in cellular respiration and ATP hydrolysis or synthesis in conjunction with self-renewal and differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The second aim will involve an investigation of mechanisms that regulate mitochondrial homeostasis, mass accumulation, and function during stem cell self-renewal and differentiation. A key component of this goal will be to determine which mitochondrial functions are most important for supporting self-renewal, pluripotency, survival, and differentiation. Reviewers were enthusiastic about this proposal’s innovative focus on a poorly understood aspect of stem cell biology. The project has important potential for providing a novel assessment of stem cell quality and for revealing key cellular properties that influence stem cell fate. In particular, the proposed study has the potential to characterize the mitochondrial target gene transcriptome in hESCs and iPSCs under a variety of conditions, and this will likely be a valuable resource to understand how mitochondrial biogenesis is modulated in the pluripotent vs. the differentiated state. Although there was some reservation about whether mitochondrial function drives cell fate decisions (as opposed to changing in response to such decisions), reviewers acknowledged the value of the proposed study for understanding relationships between cellular energetics and cell fate. The reviewers found the proposed study to be feasible and well planned. The experimental plan is carefully designed to yield meaningful results, highlights potential pitfalls, and provides alternative approaches should difficulties be encountered. There was some concern that the investigation was too exploratory and expansive, but reviewers agreed that the project has achievable milestones and is based on sound experimental approaches. Reviewers found the preliminary data to be compelling and highly supportive of the proposed study. The principal investigator (PI) is an expert on mitochondria and cellular energetics. His/her track record of publication in the stem cell field is limited, but the quality of the preliminary data demonstrates that the applicant has the appropriate expertise to carry out the proposed research. The assembled research team is outstanding, and the research environment is excellent. In summary, this proposal addresses the role of mitochondria in stem cell proliferation, maintenance of pluripotency, and differentiation potential. Despite some concerns about the broad scope of the planned research, the reviewers were extremely enthusiastic about the PI’s qualifications, the excellent research team, and the proposal’s emphasis on an important, overlooked biological issue.