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