The EMBO Journal (2011) 30, 4860–4873 | & 2011 European Molecular Biology Organization | All Rights Reserved 0261-4189/11 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells Jin Zhang1, Ivan Khvorostov1, active mitochondria and require UCP2 repression for full Jason S Hong1, Yavuz Oktay2, differentiation potential. Laurent Vergnes3, Esther Nuebel2, The EMBO Journal (2011) 30, 4860–4873. doi:10.1038/ Paulin N Wahjudi4, Kiyoko Setoguchi1, emboj.2011.401; Published online 15 November 2011 Geng Wang2, Anna Do1, Hea-Jin Jung1, Subject Categories: development; cellular metabolism J Michael McCaffery5, Irwin J Kurland6, Keywords: differentiation; metabolism; mitochondria; stem cell Karen Reue3,7, Wai-Nang P Lee4, Carla M Koehler2,7,* and Michael A Teitell1,7,8,* 1Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA, 2Department of Chemistry and Introduction Biochemistry, University of California, Los Angeles, CA, USA, 3Department of Human Genetics, University of California, Los Angeles, A distinguishing feature of human pluripotent stem cells 4 CA, USA, Department of Pediatrics, LA Biomedical Research Institute, (hPSCs) compared with differentiated cells is the capacity Torrance, CA, USA, 5Department of Biology, Integrated Imaging Center, Johns Hopkins University, Baltimore, MD, USA, 6Department of for self-renewal to maintain pluripotency. Self-renewal is Medicine, Stable Isotopes and Metabolomics Core Facility, Albert supported by unique chromatin modifications (Meshorer Einstein College of Medicine Diabetes Center, Bronx, NY, USA, and Misteli, 2006) and a regulatory circuit comprised of 7 Molecular Biology Institute, University of California, Los Angeles, CA, OCT4, NANOG, and SOX2 transcription factors (Boyer et al, USA and 8Broad Stem Cell Research Center, Jonsson Comprehensive 2005). hPSCs proliferate relatively fast, with shortened cell Cancer Center, Center for Cell Control, and California NanoSystems Institute, University of California, Los Angeles, CA, USA cycle times and a higher proportion of cells in S phase compared with most differentiated cell types (Becker et al, It has been assumed, based largely on morphologic 2006). Differences in energy status and biosynthesis from evidence, that human pluripotent stem cells (hPSCs) distinct physiologies also distinguish hPSCs from differen- contain underdeveloped, bioenergetically inactive mito- tiated cells, although the extent and regulation of these chondria. In contrast, differentiated cells harbour a differences is unknown. Thus, metabolism could provide a branched mitochondrial network with oxidative phos- relatively unexplored distinguishing feature between hPSCs phorylation as the main energy source. A role for mito- and differentiated cells. chondria in hPSC bioenergetics and in cell differentiation Mitochondria are central organelles in carbohydrate, lipid, therefore remains uncertain. Here, we show that hPSCs and amino-acid metabolism. Studies suggest that there are have functional respiratory complexes that are able to few, rounded, and non-fused mitochondria with underdeve- consume O2 at maximal capacity. Despite this, ATP gen- loped cristae in human or mouse embryonic stem cells (ESCs) eration in hPSCs is mainly by glycolysis and ATP is (St John et al, 2005; Cho et al, 2006; Chung et al,2010; consumed by the F1F0 ATP synthase to partially maintain Prigione et al, 2010) Also, hPSCs produce more lactate than hPSC mitochondrial membrane potential and cell viability. differentiated cells (Chung et al, 2010; Prigione et al, 2010), Uncoupling protein 2 (UCP2) plays a regulating role in suggesting that glycolysis supercedes oxidative phosphoryla- hPSC energy metabolism by preventing mitochondrial tion (OXPHOS) and that hPSC mitochondria may be metabo- glucose oxidation and facilitating glycolysis via a substrate lically inactive. However, this important assumption has not shunting mechanism. With early differentiation, hPSC been thoroughly tested and it remains unclear how glycolysis proliferation slows, energy metabolism decreases, and and respiration contribute to the hPSC metabolic profile. UCP2 is repressed, resulting in decreased glycolysis and Uncoupling proteins (UCPs) are mitochondrial inner mem- maintained or increased mitochondrial glucose oxidation. brane proteins that regulate cell metabolism (Nicholls and Ectopic UCP2 expression perturbs this metabolic transition Rial, 1999; Klingenberg and Echtay, 2001; Brand and Esteves, and impairs hPSC differentiation. Overall, hPSCs contain 2005). UCP1 mediates proton movement from the mitochon- drial intermembrane space to the matrix, which uncouples electron transport from ATP synthesis in brown fat to gen- *Corresponding author. CM Koehler, Department of Chemistry and erate heat (Klingenberg and Huang, 1999). In contrast, the Biochemistry, University of California, Los Angeles, 607 Charles E. function(s) of the widely expressed UCPs, including UCP2 Young Drive East, Los Angeles, CA 90095, USA. Tel.: þ 1 310 794 4834; and UCP3, are still controversial (Brand and Esteves, 2005; Fax: þ 1 310 206 4038; E-mail: [email protected] or MA Teitell, Department of Pathology and Laboratory Medicine, University of Bouillaud, 2009). UCP2 and UCP3 show uncoupling activity California, Los Angeles, 675 Charles E. Young Drive South, Los Angeles, with in vitro proteoliposome assays (Echtay et al, 2001) or CA 90095, USA. Tel.: þ 1 310 206 6754; Fax: þ 1 310 267 0382; when activated by fatty acids and free radical-derived alke- E-mail: [email protected] nals (Considine et al, 2003; Brand et al, 2004a, b). However, Received: 27 July 2011; accepted: 14 October 2011; published online: UCP2 and UCP3 have not shown physiological uncoupling 15 November 2011 activity (Cadenas et al, 2002; Couplan et al, 2002). Instead, 4860 The EMBO Journal VOL 30 | NO 24 | 2011 &2011 European Molecular Biology Organization UCP2 regulates hPSC metabolism and differentiation J Zhang et al A HSF1 HIPS2 NHDF H1 HIPS18 MitoTracker Red CMXRos (Δψ-sensitive) OCT4 (nuclear pluripotency marker) B C 1.6 1.2 HSF1 H1 HIPS2 HIPS18 NHDF 0.8 OCT4 0.4 TOM40 Citrate synth. act. 0 TIM23 H1 TOM40, TIM23- HSF1 NHDF HIPS2 HIPS18 mitochondrial proteins HEK293 Figure 1 hPSC mitochondrial morphology and abundance. (A) A fragmented hPSC mitochondrial network is shown by confocal microscopy. (B) Ratio of citrate synthase enzyme activity to total cellular protein is plotted, normalized to 1.0 for HSF1. Data are expressed as mean±s.d. (n ¼ 3). (C) Representative western blot with 50 mg protein loaded per lane. studies show that UCP2 augments fatty acid or glutamine fibroblast mitochondria were assessed for changes with oxidation and decreases glucose-derived pyruvate oxidation differentiation or reprogramming. Confocal microscopy with in mitochondria (Pecqueur et al, 2008, 2009; Bouillaud, 2009; MitoTracker Red CMXRos showed that the mitochondria in Emre and Nubel, 2010). UCP2 blocking of pyruvate entry into hESCs (HSF1 and H1 lines), and to a slightly lesser extent in the tricarboxylic acid (TCA) cycle has been postulated but not hIPSCs (HIPS2 and HIPS18 lines), are punctate, or fragmen- experimentally validated as a mechanism for enhancing ted, compared with the well-developed filamentous network aerobic glycolysis, consistent with UCP2 expression mainly of normal human dermal fibroblasts (NHDFs, also called in glycolytic tissues (Pecqueur et al, 2001) and in glycolysis- fibroblasts) (Figure 1A). hESCs differentiated by bFGF switched cancer cells (Pecqueur et al, 2001; Samudio et al, withdrawal also establish a filamentous mitochondrial net- 2008, 2009; Ayyasamy et al, 2011). Therefore, UCP2 could be work within days (Supplementary Figure S1A). Notably, a gatekeeper for the oxidation of carbon substrates, such as hIPSC lines HIPS2 and HIPS18 were reprogrammed from glucose. Notably, no role has been described for UCPs in NHDFs (Lowry et al, 2008), indicating that mitochondrial hPSC bioenergetics to date. Here, we evaluated bioenergetics morphology is reversible with de-differentiation. Combined, in human ESCs (hESCs), human-induced pluripotent stem the data show that mitochondrial fusion–fission and network cells (hIPSCs), and differentiated cells. We report an impor- morphology reflect the extent of cell differentiation. tant role for UCP2 in regulating hPSC energy metabolism and The F1F0 ATP synthase appears to play a scaffolding role the fate of early differentiating hPSCs. for the mitochondrial inner membrane cristae structure and impacts OXPHOS activity and cellular ATP levels (Giraud et al, 2002; Paumard et al, 2002; Minauro-Sanmiguel et al, Results 2005; Buzhynskyy et al, 2007; Campanella et al, 2008; Strauss A conserved mitochondrial mass ratio for hPSCs and et al, 2008). Therefore, the cristae structure of hPSCs was differentiated cells examined as a potential indicator of mitochondrial function. The distribution, abundance, fusion–fission status, and cris- Transmission electron microscopy shows that hPSC cristae tae structure of mitochondria regulates O2 consumption, are swollen, circular, and disorganized compared with the bioenergetics, apoptosis, and autophagy (Frank et al,2001; linear, stacked cristae observed in many differentiated cell Narendra et al, 2008; Chan et al, 2010; Sauvanet et al, 2010). types, including fibroblasts (Supplementary Figure S1B). Therefore, the morphology and abundance of hPSC and These features are similar