Year 2

Stem cell quality and safety for 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 this 2-year CIRM Seed Grant, we developed new approaches for analyzing respiration (oxygen consumption that drives energy production) in hESCs and hIPSCs in a series of 4 invited publications for the stem cell scientific community (; 2008). We showed that mitochondria are capable of respiring and utilizing oxygen for energy generation but do this at a very limited level compared to mature tissue cells of an adult. We speculate that this is because the cells from which hESC are derived exist physiologically in a low oxygen environment and require a switch to be turned on to facilitate oxygen consumption during development. We are working hard on understanding this switch and believe we have one of the components identified. We showed that mitochondria in reprogrammed hIPSCs are not completely reset to the embryonic state seen in hESCs, which may have implications for the use of hIPSCs in regenerative medicine. A manuscript describing the function of hESC and hIPSC mitochondria in low oxygen tension (hypoxia), in normoxia (room air), and during differentiation is being prepared for manuscript submission. 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- this work is being continued under a new funding mechanism from CIRM. 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. They may also find utility in interrogating hESC and hIPSC mitochondria function to pick the best stem cell lines for developing future cellular therapies.