Mitochondrial Metabolism in hESC and hiPSC Differentiation, Reprogramming, and Cancer

Mitochondrial Metabolism in hESC and hiPSC Differentiation, Reprogramming, and Cancer

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01397
Award Value: 
$1,323,029
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Status: 
Closed
Public Abstract: 
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.
Progress Report: 

Year 1

Great progress has been made in determining how mitochondria function in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in comparison to differentiated derivative cells, such as fibroblasts, and cancer cells. It has been assumed without much data, based largely on overall appearance under the microscope, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbor a mature mitochondrial network, with oxidative phosphorylation (OXPHOS) as the main energy source. A role for mitochondria in hPSC bioenergetics therefore remained uncertain. In just completed work funded by this CIRM Basic Biology I grant (RB1-01397), we have shown that hPSC mitochondria have functional respiration complexes that consume oxygen, which is inconsistent with the notion that hPSC mitochondria are non-functional. Despite this, energy generated in hPSCs is mainly by mechanisms that are independent of mitochondria. To help maintain intact hPSC mitochondria and overall cell viability, energy from imported glucose is burned rather than produced within mitochondria, forming an overall unusual pattern of energy utilization in hPSCs compared with differentiated cells. Combined, our data show that hPSC mitochondria are energetically functional and suggest a key mechanism(s) remaining to be discovered that converts this unique form of hPSC bioenergetics to oxygen consumption-coupled energy production within mitochondria during differentiation. Results of this work are currently under submission for publication.

Year 2

Over the past few years there have been scattered reports on the underdeveloped morphological appearance and the fragmented, perinuclear localization of mitochondria in human and mouse embryonic stem cells (hESCs), and recently in reprogrammed human induced pluripotent stem cells (hiPSCs). Based mainly upon these observations, numerous investigators have suggested that mitochondria are bioenergetically inactive or dormant in pluripotent stem cells (PSCs). This view implies that mitochondria somehow begin to function in generating cellular energy at an undefined point during differentiation in a culture dish or within the female reproductive tract. However, this conclusion was not rigorously examined, which is an important omission since so much attention is being placed on the potential for stem cells in regenerative medicine and because, at a fundamental level, it is important to understand how mitochondria, respiration, and glycolysis participate in energy production throughout mammalian development. Very surprisingly, we determined that human PSCs (both hESCs and hiPSCs) contain mitochondria that, while they do appear underdeveloped with a fragmented morphology and disorganized inner membrane, actively consume oxygen without generating much ATP. Our data shows that the mitochondrial electron transport chain complexes are assembled and functional and show that they are quantitatively equivalent in amount and functional potential to normal human dermal fibroblasts (NHDFs). Furthermore, hPSCs consume oxygen at the same rate as NHDFs, although NHDFs have a higher oxygen consumption capacity than hPSCs, which are at their maximum. NHDFs also use the electron transport chain to generate ATP by oxidative phosphorylation (OXPHOS), whereas hPSCs do not. Because of this, hPSCs rely on glycolysis for energy production. Critically, we also have generated data showing that hPSCs forced to generate ATP by OXPHOS in limiting glucose and abundant oxygen fail to do so and instead stall in the cell cycle, unlike differentiated NHDFs which adapt rapidly. This indicates that the pattern of metabolism in hPSCs is “hardwired” and a unique property of the pluripotent state, much like the unique epigenetic and transcription factor profiles that support genetic “stemness”. To maintain viability through support of the mitochondrial membrane potential, hPSCs, unlike NHDFs, hydrolyze glycolytic ATP in the mitochondrial electron transport chain complex V, also called the F1F0 ATP synthase. In fact, when mitochondrial inhibitory factor-1 (IF1), a natural inhibitor of ATP hydrolysis, is ectopically expressed in hPSCs, stem cell proliferation is slowed and viability compromised. This data suggests that hPSCs contain functional mitochondria poised for differentiation and exposure to higher, potentially toxic levels of oxygen in the female reproductive tract as development proceeds, rather than what was assumed to be a developmental switch to make PSC mitochondria expand disproportionately to their total cellular mass and become functional with differentiation. This form of metabolism is similar to, yet distinct in several ways, from the metabolism observed in many cancer cells through the well known Warburg effect. We also have generated data showing that this unique pattern of hPSC metabolism is at least partially regulated by the expression of a specific nuclear-encoded, mitochondria-imported protein, UCP2. Our mechanistic studies have generated data showing that UCP2 helps to limit ATP generation by OXPHOS in hPSCs by inhibiting pyruvate access to the TCA cycle, which reduces oxygen consumption and limits the production of reactive oxygen species. We speculate that this novel pattern of pluripotent stem cell metabolism may also regulate hPSC differentiation potential and also possibly provide a barrier to limit reprogramming efficiency to hiPSCs. This work, funded by CIRM RS1-00313, CIRM RB1-01397, CIRM TG2-01169, and by the Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research at UCLA, is in press: Zhang, J., Khvorostov, I., Hong, J.S., Oktay, Y., Vergnes, L., Nuebel, E., Wahjudi, P.N., Setoguchi, K., Wang, G., Do, A., Jung, H.-J., McCaffery, J.M., Kurland, I.J., Reue, K., Lee, W.N.P., Koehler, C.M., and Teitell, M.A. UCP2 Regulates Energy Metabolism and Differentiation Potential of Human Pluripotent Stem Cells. In press, EMBO Journal, 2011

Year 3

Over the past few years there have been scattered reports on the underdeveloped morphological appearance and the fragmented, perinuclear localization of mitochondria in human and mouse embryonic stem cells (hESCs), and recently in reprogrammed human induced pluripotent stem cells (hiPSCs). Based mainly upon these observations, numerous investigators have suggested that mitochondria are bioenergetically inactive or dormant in pluripotent stem cells (PSCs). This view implies that mitochondria somehow begin to function in generating cellular energy at an undefined point during differentiation in a culture dish or within the female reproductive tract. However, this conclusion was not rigorously examined, which is an important omission since so much attention is being placed on the potential for stem cells in regenerative medicine and because, at a fundamental level, it is important to understand how mitochondria, respiration, and glycolysis participate in energy production throughout mammalian development. Very surprisingly, we determined that human PSCs (both hESCs and hiPSCs) contain mitochondria that, while they do appear underdeveloped with a fragmented morphology and disorganized inner membrane, actively consume oxygen without generating much ATP. Our data shows that the mitochondrial electron transport chain complexes are assembled and functional and show that they are quantitatively equivalent in amount and functional potential to normal human dermal fibroblasts (NHDFs). Furthermore, hPSCs consume oxygen at the same rate as NHDFs, although NHDFs have a higher oxygen consumption capacity than hPSCs, which are at their maximum. NHDFs also use the electron transport chain to generate ATP by oxidative phosphorylation (OXPHOS), whereas hPSCs do not. Because of this, hPSCs rely on glycolysis for energy production. Critically, we also have generated data showing that hPSCs forced to generate ATP by OXPHOS in limiting glucose and abundant oxygen fail to do so and instead stall in the cell cycle, unlike differentiated NHDFs which adapt rapidly. This indicates that the pattern of metabolism in hPSCs is “hardwired” and a unique property of the pluripotent state, much like the unique epigenetic and transcription factor profiles that support genetic “stemness”. To maintain viability through support of the mitochondrial membrane potential, hPSCs, unlike NHDFs, hydrolyze glycolytic ATP in the mitochondrial electron transport chain complex V, also called the F1F0 ATP synthase. In fact, when mitochondrial inhibitory factor-1 (IF1), a natural inhibitor of ATP hydrolysis, is ectopically expressed in hPSCs, stem cell proliferation is slowed and viability compromised. This data suggests that hPSCs contain functional mitochondria poised for differentiation and exposure to higher, potentially toxic levels of oxygen in the female reproductive tract as development proceeds, rather than what was assumed to be a developmental switch to make PSC mitochondria expand disproportionately to their total cellular mass and become functional with differentiation. This form of metabolism is similar to, yet distinct in several ways, from the metabolism observed in many cancer cells through the well known Warburg effect. We also have generated data showing that this unique pattern of hPSC metabolism is at least partially regulated by the expression of a specific nuclear-encoded, mitochondria-imported protein, UCP2. Our mechanistic studies have generated data showing that UCP2 helps to limit ATP generation by OXPHOS in hPSCs by inhibiting pyruvate access to the TCA cycle, which reduces oxygen consumption and limits the production of reactive oxygen species. We speculate that this novel pattern of pluripotent stem cell metabolism may also regulate hPSC differentiation potential and also possibly provide a barrier to limit reprogramming efficiency to hiPSCs. All of this work and much more, funded by CIRM RS1-00313, CIRM RB1-01397, CIRM TG2-01169, and by the Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research at UCLA, is either published or in press: Zhang, J., Khvorostov, I., Hong, J.S., Oktay, Y., Vergnes, L., Nuebel, E., Wahjudi, P.N., Setoguchi, K., Wang, G., Do, A., Jung, H.-J., McCaffery, J.M., Kurland, I.J., Reue, K., Lee, W.N.P., Koehler, C.M., and Teitell, M.A. UCP2 Regulates Energy Metabolism and Differentiation Potential of Human Pluripotent Stem Cells. EMBO Journal, 30:4860-4873, 2011 (commentary by L Cantley in same issue.) Zhang, J., Nuebel, E., Wisidagama, D.R.R., Setoguchi, K., Hong, J.S., Van Horn, C. M., Imam, S.S., Vergnes, L., Malone, C.S., Koehler, C.M., and Teitell, M.A. Measuring Energy Metabolism in Cultured Cells, Including Human Pluripotent Stem Cells and Differentiated Cells. Nature Protocols, 7:1068-1085, 2012 Zhang, J., Nuebel, E., Daley, G.Q., Koehler, C.M., and Teitell, M.A. Metabolism in Pluripotent Stem Cell Self-Renewal, Differentiation, and Reprogramming. Invited, in revision, Cell Stem Cell, 2012

Year 4

We determined that human pluripotent stem cells (PSCs; both hESCs and hiPSCs) contain mitochondria that, while they do appear underdeveloped with a fragmented morphology and disorganized inner membrane, actively consume oxygen without generating much ATP. Our data shows that the mitochondrial electron transport chain complexes are assembled and functional and show that they are quantitatively equivalent in amount and functional potential to normal human dermal fibroblasts (NHDFs). Furthermore, hPSCs consume oxygen at the same rate as NHDFs, although NHDFs have a higher oxygen consumption capacity than hPSCs, which are at their maximum. NHDFs also use the electron transport chain to generate ATP by oxidative phosphorylation (OXPHOS), whereas hPSCs do not. Because of this, hPSCs rely on glycolysis for energy production. Critically, we also have generated data showing that hPSCs forced to generate ATP by OXPHOS in limiting glucose and abundant oxygen fail to do so and instead stall in the cell cycle, unlike differentiated NHDFs which adapt rapidly. This indicates that the pattern of metabolism in hPSCs is “hardwired” and a unique property of the pluripotent state, much like the unique epigenetic and transcription factor profiles that support genetic “stemness”. To maintain viability through support of the mitochondrial membrane potential, hPSCs, unlike NHDFs, hydrolyze glycolytic ATP in the mitochondrial electron transport chain complex V, also called the F1F0 ATP synthase. In fact, when mitochondrial inhibitory factor-1 (IF1), a natural inhibitor of ATP hydrolysis, is ectopically expressed in hPSCs, stem cell proliferation is slowed and viability compromised. This data suggests that hPSCs contain functional mitochondria poised for differentiation and exposure to higher, potentially toxic levels of oxygen in the female reproductive tract as development proceeds, rather than what was assumed to be a developmental switch to make PSC mitochondria expand disproportionately to their total cellular mass and become functional with differentiation. This form of metabolism is similar to, yet distinct in several ways, from the metabolism observed in many cancer cells through the well known Warburg effect. We also have generated data showing that this unique pattern of hPSC metabolism is at least partially regulated by the expression of a specific nuclear-encoded, mitochondria-imported protein, UCP2. Our mechanistic studies have generated data showing that UCP2 helps to limit ATP generation by OXPHOS in hPSCs by inhibiting pyruvate access to the TCA cycle, which reduces oxygen consumption and limits the production of reactive oxygen species. We speculate that this novel pattern of pluripotent stem cell metabolism may also regulate hPSC differentiation potential and also possibly provide a barrier to limit reprogramming efficiency to hiPSCs. All of this work and much more, funded by CIRM RS1-00313, CIRM RB1-01397, CIRM TG2-01169, and by the Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research at UCLA, is either published or in press: Zhang, J., Khvorostov, I., Hong, J.S., Oktay, Y., Vergnes, L., Nuebel, E., Wahjudi, P.N., Setoguchi, K., Wang, G., Do, A., Jung, H.-J., McCaffery, J.M., Kurland, I.J., Reue, K., Lee, W.N.P., Koehler, C.M., and Teitell, M.A. UCP2 Regulates Energy Metabolism and Differentiation Potential of Human Pluripotent Stem Cells. EMBO Journal, 30:4860-4873, 2011 (commentary by L Cantley in same issue.) Zhang, J., Nuebel, E., Wisidagama, D.R.R., Setoguchi, K., Hong, J.S., Van Horn, C. M., Imam, S.S., Vergnes, L., Malone, C.S., Koehler, C.M., and Teitell, M.A. Measuring Energy Metabolism in Cultured Cells, Including Human Pluripotent Stem Cells and Differentiated Cells. Nature Protocols, 7:1068-1085, 2012 Zhang, J., Nuebel, E., Daley, G.Q., Koehler, C.M., and Teitell, M.A. Metabolism in Pluripotent Stem Cell Self-Renewal, Differentiation, and Reprogramming. Cell Stem Cell, 2:589-595, 2012 Dabir, D., Hasson, S.A., Setoguchi, K., Johnson, M.E., Wongkongkathep, P., Douglas, C.J., Zimmerman, J., Damoiseaux, R., Teitell, M.A., and Koehler, C.M. MitoBloCK-6: A Small Molecule Inhibitor of Redox-Regulated Protein Translocation in Mitochondria. Developmental Cell, 25:81-92, 2013

© 2013 California Institute for Regenerative Medicine