Control of Human Embryonic Stem Cell Self-Renewal, Pluripotency and Differentiation

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01397
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
All of the diverse cells in a human body contain the same genetic information, and originally arose from a single cell, a fertilized egg. Embryogenesis is a result of cell division followed by differential gene expression, to selectively activate only the genes needed for development of each specialized cell type. By understanding the multiple gene activities required to either maintain stem cell pluripotency or effect cell specific differentiation, it should be possible to define conditions under which undifferentiated stem cells may be grown in large volume in culture, or induced to become mature cell types of therapeutic interest. The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew or exit the pluripotent state and begin to differentiate. In preliminary studies, our laboratory has identified two protein complexes, SCC-A and SCC-B, required for the activity of genes needed for stem cell self-renewal. Here we propose to characterize the component proteins of these complexes. SCCs are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. We have also identified another protein, TAF3, that may play a central role in influencing whether stem cells self-renew or differentiate into various cell types. Understanding TAF3 function may provide a strategy to better control stem cell differentiation, a necessary step toward tissue replacement therapy. A third focus of the proposal is to understand the impact of eliminating specific stem cell gene regulatory proteins via intracellular protein degradation pathways. Our preliminary data suggest that degradation of proteins crucial to controlling gene expression in pluripotent stem cells is an important regulated step in initiating differentiation. We propose biochemical and molecular biological studies to identify protein degradation pathways involved in maintaining these gene regulatory proteins, or marking them for destruction. Similar to the SCC complexes, these proteins could provide excellent targets for drug design, to increase or decrease the ability of stem cells to divide or differentiate.
Statement of Benefit to California: 
The ultimate goal of these studies is the development of therapies for diseases that are fundamentally the result of inappropriate levels of gene expression, cell division and differentiation. The proposed experiments will demonstrate how gene activity is controlled, either to maintain a renewing population of stem cells, or to direct differentiation of specific mature cell types. Because all biological activities; cell division, differentiation and function, are the result of differential gene expression, an understanding of the gene regulatory networks controlling these processes will be crucial for drug development and testing. The proposed experiments will benefit the people of the State of California immediately by supporting the state economy. This project will directly employ three people, supporting two graduate students, and one highly trained Research Specialist. Additionally, it will support the purchase of reagents and services necessary to conduct the proposed research, indirectly contributing to the employment of many other individuals in the biotechnology, education and service industries. In the longer term, this research will benefit the people of California by helping our state to maintain its status as a major force in biomedical research. A thorough understanding of basic stem cell biology will be necessary to support the ultimate development of safe and effective therapeutic applications, and the research efforts toward this end will contribute to the continued success of the outstanding universities and robust biomedical research community that have made California a leader in the fields of biotechnology and medicine. The work described in this proposal will likely reveal gene activities that are essential for the establishment, survival and maintenance of stem cells and differentiated cell populations. This knowledge may potentially identify previously unknown drug targets that will allow the screening of novel classes of pharmeceuticals, or to allow stem cells to be grown in culture as large volumes of homogeneous cells, a necessary prelude to their use in stem cell therapy. Understanding the role of coordinated turnover of transcriptional regulators during stem cell differentiation may also represent a promising new strategy to predictably influence the pluripotent state and differentiation program in stem cells.
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