The molecular mechanism of epigenetic reprogramming by defined factors

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01397
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
Epigenetic reprogramming of a somatic cell into pluripotent stem cells has raised enormous interest in the scientific community because of the multitude of applications in basic biology and clinical research. Particularly important are the impacts on regenerative medicine, as reprogramming would allow the generation of patient-derived (and thus individualized) stem cells that could be used to differentiate into functional donor cells for transplantation therapies. For many years epigenetic reprogramming was only achievable in animals through somatic cell nuclear transfer (SCNT) into enucleated oocytes, a technically and logistically challenging and very inefficient procedure. To this day, epigenetic reprogramming through SCNT has not been successful in human despite great efforts. In August 2006, Shinya Yamanaka has reported for the first time that retroviral overexpression of 4 transcription factors is sufficient to induce pluripotent stem (iPS) cells from mouse fibroblasts. Similarly to the mouse cells, defined factor-induced reprogramming was then shown to work also in human cells. This paved the way for potential clinical application as for the first time patient-specific stem cells were created. However, in both mouse and human systems the efficiency of reprogramming is extremely low. This indicates that scientists have to rely on uncontrolled events leading to reprogramming. Before we will be able to use these iPS cells in a clinical setting we will therefore have to have a much better knowledge of the reprogramming process to be able to improve the efficiency. Once we know exactly what is going on in these reprogramming cells, have absolute control over the process and can reach efficiencies close to 100%, the generation of human iPS cells from somatic cells will very likely be a safe procedure. This research proposal addresses some fundamental questions why the reprogramming process is so inefficient. In this study we propose to optimize the expression of the reprogramming factors, to identify the best donor cell population for reprogramming, and finally to attempt to provide a list of new factors that can improve the reprogramming efficiency.
Statement of Benefit to California: 
The benefits of this research to California are two fold: 1) it stimulates the economy by directly creating 5 jobs or salary support for California citizens and supporting California business because of regular purchase of consumables and research equipment. 2) At the same time the money is spent to support a research project that aims to improve the development of a novel type of pluripotent stem cells that could be used to treat a variety of diseases such as neurodegenerative diseases, diabetes, spinal chord injury, and genetic skin and muscle diseases. The impact of such a novel stem cell therapy on the California health system would be enormous. The number of patients suffering from neurodegenerative diseases alone are stunning: Currently 1-2% of the population older than 65 years is diagnosed with Parkinson's disease a devastating disease affecting cognition and body movements and 20% of people older than 75 years suffer from Alzheimer's disease a progredient neurodegenerative disease leading to loss of higher cognitive functions. Any progress towards relieving the major symptoms of patients suffering from these neurodegenerative diseases will be of immense benefit to the State of California and its citizens. In addition, all the tools and reagents that we develop will be made widely available to Californian researchers and we have selected a California-based company for potential commercialization. We hope that California-based physicians will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Progress Report: 
  • 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.
  • 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
  • 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
  • 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