Mechanisms of Lineage Commitment in Human Neural Crest Stem Cells

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01397
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
Diseases arising from defects in NC specification, migration and differentiation referred to as neurocristopathies (total ~74) include various skeletal dysmorphology syndromes (e.g. Apert and Beare-Stevenson cutis gyrate syndromes), diseases of the nervous system (neurofibromatosis and Hirschsprung’s disease, peripheral neuropathies such as Familial Dysautonomia) and pigment disorders (Waardenburg syndrome). The defect is often confined to one or two NC lineages, as in pigment abnormalities and the treatment of PNS disorders will require a uniform population of lineage-committed progenitors. In addition, in vitro applications such as initial screening for drug toxicity will require large quantities of pure NC progenitors. In particular, derivation of functional chondrocytes may provide a much-needed alternative for cartilage regeneration. Recently, by means of the support from a CIRM Seed grant, we generated a uniform population of neural crest stem cells (NCSC). These human NCSC can differentiate in vitro producing all neural crest lineages including sensory and autonomic neurons, Schwann cells, smooth muscle cells, melanocytes, adipocytes and chondrocytes. We propose comprehensive in vitro analysis of differentiation in NC for the following two reasons: 1. All previous evidence was obtained in the model organisms. No studies investigating the role of transcription factors or extracellular matrix (ECM) in human neural crest self-renewal and differentiation were reported to date. 2. It is possible that unsuspected conditions will promote directed differentiation of human NCSC compared to physiological stimuli. Specifically we will: 1. Identify transcription factors regulating human NCSC differentiation using a functional genomic approach; 2. Investigate the role of various matrix components and their elasticity in human NCSC differentiation. The deliverables will include: first, a verified set of TFs that regulate human NCSC lineage commitment and differentiation; second, a knowledge of how the nature and stiffness of various ECM matrices affects differentiation into neural crest lineages. The knowledge, tools and reagents will be available to other academic researchers and commercial entities. If successful, the proposed approach will help us to understand mechanisms of NC differentiation along specific NC lineages. This knowledge will allow the development of tools and reagents for diagnostic and therapeutic applications such as screening assays for drugs affecting human sensory neurons, neuronal replacement for peripheral neuropathies, and the generation of functional Schwann cells.
Statement of Benefit to California: 
Diseases arising from defects in NC specification, migration and differentiation referred to as neurocristopathies (total ~74) include various skeletal dysmorphology syndromes (e.g. Apert and Beare-Stevenson cutis gyrate syndromes), diseases of the nervous system (neurofibromatosis and Hirschsprung’s disease, peripheral neuropathies such as Familial Dysautonomia) and pigment disorders (Waardenburg syndrome). The defect is often confined to one or two NC lineages, as in pigment abnormalities and the treatment of PNS disorders will require a uniform population of lineage-committed progenitors. We propose comprehensive in vitro analysis of differentiation in NC for the following two reasons: 1. All previous evidence was obtained in the model organisms. No studies investigating the role of transcription factors or extracellular matrix (ECM) in human neural crest self-renewal and differentiation were reported to date. 2. It is possible that unsuspected conditions will promote directed differentiation of human NCSC compared to physiological stimuli. The deliverables will include: first, a verified set of TFs that regulate human NCSC lineage commitment and differentiation; second, a knowledge of how the nature and stiffness of various ECM matrices affects differentiation into neural crest lineages. The knowledge, tools and reagents will be available to other academic researchers and commercial entities. If successful, the proposed approach will help us to understand mechanisms of NC differentiation along specific NC lineages. This knowledge will allow the development of tools and reagents for diagnostic and therapeutic applications such as screening assays for drugs affecting human sensory neurons, neuronal replacement for peripheral neuropathies, and the generation of functional Schwann cells. An effective, straightforward, and understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. In addition, this program and its many complementary programs will generate potentially very large, tangible monetary benefits to the citizens of California. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by the CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.
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