348 Research Article

IntroductionHuman embryonic stem cells (hESCs) are long-term self-renewingcells that are able to differentiate into any cell type in the body(Thomson et al., 1998). The potential of hESCs to act as anunlimited source for somatic cell generation is of major interest fordownstream applications, including drug testing, disease modellingand cell replacement therapy (Braam et al., 2009; Desbordes et al.,2008; Han et al., 2009; Kiskinis and Eggan, 2010; Passier et al.,2008; Zeng and Rao, 2008). Additionally, these cells might yieldinsights into the processes underlying cellular senescence, cancerand ageing (Zeng, 2007). Obtaining a more complete understandingof the biology of pluripotent cells is important for maximising thebenefits from these areas.

The cells of the inner cell mass of the blastocyst, from whichhESCs are derived, divide rapidly. This property of aggressivegrowth is reflected in cultured ESCs by their ability to formteratomas and their short-term growth-factor-autonomous cellcycling (Becker et al., 2010; Blum and Benvenisty, 2009), althoughthere are differences in the division rate of different pluripotent celltypes (Hanna et al., 2010). Progenitor cells in the adult organism,such as multipotent neural stem cells (NSCs), must possess theunique properties of long-term viability and self-maintenance,while remaining in what is typically a quiescent state. MultipotentNSCs can be derived in pure populations from cultured hESCs(Reubinoff et al., 2001; Swistowski et al., 2009; Zhang et al.,2001), but little is known about their bioenergetic requirements orhow closely their energy metabolism is governed by the constraints

of the genetic developmental programme. The transition from apluripotent stem cell through progressive stages of differentiationprobably involves dynamic changes in the energy demand forcellular processes, depending on the needs of the individual celltypes. ATP generation in aerobic cells is divided between two mainpathways: anaerobic glycolysis and mitochondrial oxidativephosphorylation. On the basis of changing energy demands, andthe need to limit the production of mitochondrial reactive oxygenspecies, it might be expected that the relative contribution of thesetwo pathways would change during differentiation.

Some models suggest that an expansion of the mitochondrialpopulation regardless of the differentiation lineage might be auniversal phenomenon from the onset of ESC differentiation(Facucho-Oliveira and St John, 2009). However, many signallingand energy-consuming processes might be decreased in the earlytransition to a differentiated state, and it might not be expected thatthis would be accompanied by a mitochondrial expansion(Armstrong et al., 2006; Becker et al., 2006; Efroni et al., 2008;Meshorer and Misteli, 2006). By contrast, the evidence thatmitochondrial biogenesis occurs in some instances of somaticstem-cell differentiation could be consistent with the acquisitionof specialised energy-requiring processes, as reported formesenchymal stem-cell differentiation (Chen et al., 2008; Uldry etal., 2006). Changes in mitochondrial regulation might also bedriven by genetic developmental programmes, reflecting the typicalenergy requirements of these cell types in vivo as opposed to theenergy requirements in culture. This provides a rationale for

SummaryHere, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derivedneural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATPsupply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% oftheir ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both inabsolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs isconsistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor g coactivator(PGC)-1, PGC-1 and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types.Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higherrate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media.Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somaticcell types, and supports important roles for mitochondria and secretion in hESC biology.

Key words: Mitochondria, Pluripotency, Embryonic stem cells, Neural stem cells, Differentiation, Secretion

Accepted 10 October 2010Journal of Cell Science 124, 348-358 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.072272

A reduction in ATP demand and mitochondrial activitywith neural differentiation of human embryonic stemcellsMatthew J. Birket, Adam L. Orr, Akos A. Gerencser, David T. Madden, Cathy Vitelli, Andrzej Swistowski,Martin D. Brand and Xianmin Zeng*Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945, USA*Author for correspondence (xzeng@buckinstitute.org)

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following the genetic regulators of energy metabolism in concertwith the bioenergetics to illuminate the different forces drivingchanges in mitochondrial function.

In the present study, we characterise mitochondrial function inhESCs and in several stages of neuronal differentiation usingadvanced bioenergetic technologies. We conclude that hESCs havea high mitochondrial activity that declines disproportionately,alongside several related parameters of mitochondrial biogenesis,with differentiation into NSCs and that this is coupled to a decreasein overall ATP turnover, a decrease in macromolecule biosynthesisand a decrease in protein secretion.

ResultshESCs have a high rate of energy turnoverTo study the bioenergetics of hESC neural differentiation, weexamined hESCs (Fig. 1Ai), hESC-derived NSCs (Fig. 1Aii) andNSCs differentiated for 35 days, which consist of >80% -III-tubulin-positive neurons (termed D-35; Fig. 1Aiii), as shownpreviously (Swistowska et al., 2010; Swistowski et al., 2009). Ithas been demonstrated previously that these NSCs are nestin- andSox1-positive and have a wide differentiation capacity (Swistowskiet al., 2009). For comparisons with another somatic cell type, wechose to examine a well-characterised primary human fetal-foreskin-derived fibroblast line (BJ; termed HDF).

The oxygen consumption rate and extracellular acidificationrate were determined in adherent cultures using a Seahorse XF-24extracellular flux analyzer, as described previously (Gerencser etal., 2009). Basal respiration rate in hESC colonies was 1.8-foldhigher than in NSCs and 2.8-fold higher than in HDFs, whennormalised to the cell protein level (Fig. 1B). The coupled rate (thetotal basal rate minus the rate with oligomycin, where oligomycin

349Bioenergetics of human embryonic stem cells and neural stem cells

is an inhibitor of ATP synthesis) was 111 pmol of O2 per minuteper 10 g of cell protein in hESCs and dropped to 54 pmol of O2

per minute per 10 g of cell protein in NSCs, indicating that themitochondrial ATP output decreases in the transition from hESCsto NSCs. Data were combined from the I6 and H9 lines for hESCsand NSCs to increase the reliability of our final determinations (nosignificant difference was observed between these lines).Differentiation of NSCs into D-35 cells caused little change inrespiration or oxidative phosphorylation compared with the parentalNSCs. Furthermore, the maximal uncoupler-evoked oxygenconsumption rate (indicating maximal respiratory capacity) wassignificantly decreased in NSCs when compared with hESCs. Theextracellular acidification rate is mainly due to lactate andbicarbonate production and, when calibrated as the protonproduction rate, indicates glycolytic rate (Wu et al., 2007). Incontrast with respiration, the glycolytic rate was slightly increasedin NSCs but declined significantly in D-35 cells (Fig. 1C). Anabsolute quantification of both the oligomycin-sensitive oxygenconsumption rate and glycolytic rate can be converted into the ATPproduction rate, to assess the relative contribution in each cell type.The oligomycin-sensitive oxygen consumption was converted intothe ATP production rate using a P/O ratio of 2.3 (Brand, 2005), andthere is a one-to-one relationship between the lactate productionrate and the ATP production rate from glycolysis. Remarkably,hESCs made at least 77% of their ATP using oxidativephosphorylation, whereas this value was significantly lower inNSCs (55%) and in HDFs (59%) (Fig. 1D, both P<0.01 comparedwith hESCs), but was similar to that of hESCs in D-35 cells (76%).The results in Fig. 1D show that hESCs had a faster turnover ofATP than NSCs, D-35 cells or HDFs and that this demand is metprimarily by higher oxidative phosphorylation in hESCs.

Fig. 1. hESCs show high levels of mitochondrial activity and a bias for aerobic ATP production. (A)The differentiation scheme used for measurements.Pluripotent I6 hESCs (i), multipotent I6 neural stem cells (NSCs) (ii) and differentiated I6 NSCs (D-35) (iii), stained for neuronal Tuj--tubulin. BJ fibroblasts(HDF) were also analysed (image not shown). (B)Oxygen consumption rate (OCR) measured on adherent cultures normalised to cell protein levels. Basal,endogenous rate; Oligomycin, ATP-synthase-inhibited rate; FCCP, maximal uncoupled rate; and Rot + Ant A, rotenone- and antimycin-A-inhibited rate. (C)Protonproduction rate from measurements taken in tandem with respiration rates. (D)ATP production rate from oxidative phosphorylation and glycolysis. Results shownare the means+s.e.m. from independent experiments (hESC, n10; NSC, n11; D-35, n3; and HDF, n4). The statistical significance was calculated from a one-way ANOVA using Dunnett’s correction. *P<0.05, **P<0.01.

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Mitochondrial content during hESC neural differentiationTo address the apparent bias hESCs show towards high oxidativephosphorylation for ATP production, we assessed variousparameters of mitochondrial content in each cell type. Westernblots showed that although the levels of voltage-dependent anion-selective channel (VDAC) remained unchanged upon neuraldifferentiation, cytochrome c levels were markedly decreased inNSCs but were upregulated again upon differentiation into D-35cells (Fig. 2A,B). The important antioxidant proteins manganesesuperoxide dismutase (MnSOD) (mitochondrial) and copper/zincsuperoxide dismutase (CuZnSOD) (cytosolic) showed little change(MnSOD was slightly reduced in NSCs), indicating that the cellularantioxidant system remains fairly stable between these states.

Quantification of absolute mitochondrial DNA (mtDNA) copynumber revealed that hESCs are rich in mtDNA, with around1200–1500 copies per cell, whereas NSCs, D-35 cells and HDFscontain 600–1000 copies per cell (Fig. 2C; values are the means ofthe H9 and I6 cell lines at each differentiation stage). A third hESCline BG01 was found to have approximately 1200 copies per cell.No significant difference was found in mtDNA content betweenhESCs maintained on feeder cells and those maintained on matrix

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in mouse embryonic fibroblast (MEF)-conditioned medium (datanot shown). When normalised for cell size (see supplementarymaterial Fig. S1), these values equate to an approximately 1.5- to2-fold greater mtDNA density in hESCs. Differentiation of hESCs,as embryoid bodies or as a monolayer in inactivated serum-replacement-containing medium, showed that this progressivetransition in mtDNA copy number occurred during thedifferentiation process itself and that by day 12 the amount wasclose to the content of the NSCs (Fig. 2D). This pattern wasmimicked in B6 mouse ESC differentiation (supplementary materialFig. S2).

We also measured mitochondrial organelle content by employinglive-cell confocal imaging stereology in cells labelled withMitoTracker Red and calcein-AM. In agreement withdifferentiation-based changes in respiration and mtDNA content,stereology revealed a small but significant reduction inmitochondrial volume density from 5.4±0.3% in hESCs to4.0±0.3% in NSCs (Fig. 2E,F; P<0.01), which was reversed uponfurther differentiation (D-35: 5.9±0.4%). The mitochondrial volumedensity of the HDFs was similar to that of the NSCs (HDF:4.0±0.2%; P<0.01 compared with hESCs).

Fig. 2. Mitochondrial content is altered with hESC differentiation. (A)Western blots for mitochondrial [cytochrome c (Cyt C) and VDAC] and antioxidantproteins (MnSOD and CuZnSOD) in whole-cell lysates from hESC cultures with corresponding NSCs and differentiated NSCs (D-35), compared with BJfibroblasts (HDF). (B)Protein band quantification for cytochrome c and VDAC from three independent lysates for each sample normalised to the hESC proteinlevel. Results represent the means+s.d. of combined data from the I6 and H9 lines. (C)mtDNA copy number per cell in hESCs and differentiated cells. Resultsshown are the means+s.e.m. for the H9 and I6 stem cell lines and the HDFs. (D)mtDNA copy number changes during hESC differentiation as embryoid bodies oras a monolayer. Results shown are the means+s.e.m. for independent experiments in the H9 and I6 stem cell lines. (E)Confocal images for mitochondrial volumeratio determination. Calcein-AM labels the cytoplasm and MitoTracker Red (MTR) labels the mitochondria. The upper two panels show the raw images for eachchannel and the lower two panels show the binary processed images, which were used as input for the calculation. (F)Mitochondrial volume ratio quantificationfrom confocal imaging. Data represent means±s.e.m. for at least four independent experiments with both H9 and I6 stem cell lines. The statistical significance wascalculated from a one-way ANOVA using Dunnett’s correction. **P<0.01.

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Pluripotent stem cells express mitochondrial generegulators in a pattern indicative of high reliance onoxidative phosphorylationTo investigate whether the high levels of mitochondrial activityand changes in mitochondrial biogenesis upon differentiation hadunderlying nuclear genetic regulation, we examined the expressionof the ‘master regulators’ of mitochondrial gene expression – thatis transcriptional coactivators and corepressors thought to controlthe expression of multiple downstream targets (Feige and Auwerx,2007; Handschin and Spiegelman, 2006). We measured theexpression levels of the genes encoding the two transcriptionalcoactivators peroxisome-proliferator-activated receptor g coactivator(PGC)-1 and PGC-1 and the transcriptional corepressor receptor-interacting protein 140 (RIP140). We first compared hESCs withseveral somatically derived primary cell types and two cancer celllines. Consistent with our functional observations, the mRNAsencoding PGC-1 and PGC-1 were both expressed to a greaterextent in hESCs than in most of the somatic cell types (Fig. 3A,B).The cell types most similar to hESCs were melanocytes (HEMN-LP) and the cancer cell line MCF7, which showed high expressionof the mRNAs encoding both PGC-1 and PGC-1. RIP140, arepressor of genes involved in oxidative metabolism, was weaklyexpressed in hESCs compared with all the other cell types (Fig.

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3C). PGC-1 and PGC-1 expression decreased and RIP140expression increased during hESC differentiation into NSCs (Fig.3D,E). Similar changes in PGC-1 and PGC-1 expression withhESC embryoid body differentiation have also been reportedrecently (Prigione et al., 2010). A large induction in PGC-1 upondifferentiation into D-35 cells is consistent with our observation ofincreased mitochondrial biogenesis and a return to a reliance onmitochondrial ATP production in these cells.

To confirm further the expression pattern of these genes inhuman pluripotent cells, we assessed the expression of these genesin an induced pluripotent stem cell (iPSC) line derived from IMR90fibroblasts (Mali et al., 2010). There was a strong induction of themRNA encoding PGC-1, whereas that encoding RIP140 wasrepressed upon reprogramming (Fig. 3F). PGC-1 was not inducedbut already had elevated expression in these fibroblasts comparedwith the other fibroblast strains we measured. iPSCs that wereinduced to differentiate into NSCs and D-35 cells, as describedpreviously (Swistowski et al., 2010), showed similar mRNAexpression changes to the hESC differentiations (Fig. 3F).

Finally, we attempted to control the energetic supply indifferentiated cells by stable overexpression of PGC-1 or shorthairpin RNA (shRNA) depletion of RIP140 in I6 NSCs orHDFs. Surprisingly, neither manipulation substantially boosted

Fig. 3. Gene expression profiles for three metabolic master regulators highlight similarities between pluripotent stem cells and other energy demandingcell types. Real-time reverse transcription–PCR data for expression of the mRNA encoding PGC-1 (A), PGC-1 (B) and RIP140 (C) in I6 and BG01 hESC linesmaintained on feeder cells (MEF) or on Geltrex in conditioned medium (GTX) compared with seven other primary cell types and two cancer cell lines. HDF,human diploid fibroblast; HEK, human epidermal keratinocyte; HEMN-LP, human epidermal melanocyte-low pigmented; HMVEC, human mammary veinendothelial cells; HUVEC, human umbilical vein endothelial cells; HPASMC, human pulmonary artery smooth muscle cells; HMEC, human mammary epithelialcells, MCF7, breast cancer cell line; and HeLa, cervical cancer cell line. Results represent means+s.e.m. for 2–4 independent RNA preparations. (D)Real-timereverse transcription–PCR data during differentiation of hESCs as embryoid bodies or as a monolayer. Data represent the mean values for two hESC lines (H9 andI6). (E)mRNA expression for NSCs and D-35 cells relative to their parental hESC lines. (F)mRNA expression changes during reprogramming of IMR31fibroblasts into iPSCs and differentiation into iPSC-NSCs and iPSC-D35 cells.

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mitochondrial respiration or respiratory capacity (supplementarymaterial Fig. S3). RIP140 knockdown did subtly increase maximalrespiratory capacity in both cell types and stimulated glycolysis inNSCs. These findings suggest that manipulation of these genesindividually is insufficient to alter mitochondrial bioenergetics inthese cell types.

Plasma and mitochondrial membrane potentialmeasurements in hESCs, NSCs and HDFsThe higher basal ATP turnover and maximal ATP-generatingcapacity of hESCs cannot be explained solely by the abundance ofmitochondria in these cells. Mitochondrial membrane potential(m) accounts for the majority of the proton-motive force usedfor driving ATP synthesis and therefore has a significant effect onthe maximal ATP-generating capacity (Nicholls, 2006). The restingm reflects the balance between supply (substrate and terminaloxidation) and demand (ATP consumption). A high ATP demandin hESCs might outstrip supply and thus result in a lower m thanin differentiated cells such as NSCs and HDFs, where ATP demandis smaller. Conversely, a high ATP supply originating from m

might keep ATP consumption running faster in hESCs.m in adherent cultures of hESCs and related lines was

determined by fluorescence microscopy. All known m

measurement techniques (fluorescence, voltammetric or isotopic)rely on the potential-dependent distribution of lipophilic cations,such as the fluorescent indicator tetramethylrhodamine methylester (TMRM). The mitochondrial accumulation of these cationsis a function of m, the plasma membrane potential (p) and the

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mitochondrial-matrix-to-cell-volume ratio (Nicholls, 2006). Theseparameters are expected to differ between cell types. To unveilm, p was followed with a lipophilic anionic plasma membranepotential indicator (PMPI) (Nicholls, 2006), and volume ratioswere determined separately. TMRM and PMPI fluorescence ofwhole microscopic view fields were monitored in time, and p

and m were determined in millivolts by temporally deconvolutingthe fluorescence intensity time lapses, taking into account the slowpotential-dependent redistribution and nernstian behaviour of theseprobes (A.A.G. and M.D.B., unpublished).

p was calibrated by establishing a K+-equilibrium potential{a potential governed by the intra- and extra-cellular (EC) [K+]}using a cocktail of K+-channel openers and making stepwiseincrements in [K+]EC (Fig. 4A–C). The resting p pooled fromthe two hESC lines (I6 and H9), as well as from HDFs is shownin Fig. 4D. The mitochondrial-matrix-to-cell-volume ratio wasobtained as the product of the mitochondria-to-cell-volume ratio(see above) and the matrix-to-mitochondria-volume ratio. The latterwas measured as the volume density of matrix by transmissionelectron microscopic stereology and was 0.84 for hESCs and 0.76for NSCs. For HDFs, we assumed the same ratio as for NSCs.Interestingly, mitochondria in hESCs were mostly in the ‘orthodox’conformation (Hackenbrock, 1966), whereas NSCs showed a‘condensed’ conformation, with higher electron density in thematrix and more dilated cristae (Fig. 4G). The resting m wascalculated from the efflux kinetics of TMRM following an acuteand complete mitochondrial depolarisation (Fig. 4E,F). The decayof TMRM fluorescence under these conditions was determined by

Fig. 4. Resting plasma and mitochondrial membrane potential determination in hESCs and differentiated cells. (A)Time-lapse images of a hESC colonylabelled with TMRM and PMPI potentiometric probes, under basal conditions, following addition of the K+ equilibrium cocktail (KEC) and the completedepolarisation cocktail (CDC). (B)Fluorescence intensity traces corresponding to A. Following the KEC addition, sequential replacement steps with KCl-basedmedium were performed as indicated. Finally the plasma membrane was completely depolarised with the addition of the CDC. (C)Mean normalised PMPIfluorescence values obtained for the three indicated cell types at the indicated extracellular K+ concentrations. (D)Mean (+s.e.m.) resting plasma membranepotentials for hESCs, NSCs and HDFs (hESC and NSC, n8; HDF, n3). Potentials were derived from C as given in the supplementary methods. (E)Time-lapseimages of a hESC colony labelled with TMRM and PMPI, under basal conditions, 40 seconds after addition of the mitochondrial depolarisation cocktail (MDC)and after addition of the CDC. (F)Fluorescence intensity traces corresponding to E. Mitochondrial depolarisation was triggered with the MDC as indicated andfinally complete depolarisation was achieved with the CDC. Parameters derived from the fluorescence traces are shown in supplementary material Fig. S3.(G)Electron micrographs of typical mitochondria in hESCs and NSCs (viewed at 105,000�). (H)Mean (+s.e.m.) resting mitochondrial membrane potential valuesfor hESCs, NSCs and HDFs (hESC, n28; NSC, n16; and HDF, n13). Potentials were derived from experiments similar to those shown in F and as given in thesupplementary methods. The statistical significance was calculated from a one-way ANOVA using Dunnett’s correction. **P<0.01.

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the previous resting m, volume ratios and the present p (seethe supplementary methods).

The mean determinations are shown in Fig. 4H. We concludethat there is no significant change in m upon differentiation ofhESCs into NSCs. Therefore the decrease in respiration in NSCswhen compared with hESCs is not caused by a large decrease inATP supply or demand; instead supply and demand are kept inbalance by concerted regulation.

The higher ATP turnover in hESCs compared with NSCs atslower growth rates is probably explained bymacromolecule secretion in hESCsOur data suggest that hESCs turnover ATP more rapidly thandifferentiated cells. To pinpoint the process(es) responsible for theincreased ATP demand, we compiled a complete budget of the ATPdemand in hESCs and NSCs. Individual ATP-consuming processeswere acutely inhibited, causing a consequential drop in ATP demandand triggering a proportional drop in respiration. Dose–responsecurves for the protein translation inhibitor cycloheximide, the DNAand/or RNA synthesis inhibitor actinomycin D and the proteasomeinhibitor MG132 are shown in supplementary material Fig. S4. Inaddition, the tubulin and actin turnover and the activities of Na+/K+-ATPase, the endoplasmic reticulum (ER) and plasma membraneCa2+-ATPase and xanthine oxidase were investigated. Using theminimum saturating concentrations of each inhibitor, the relativecontributions to the total respiration of these cells were assignedcumulatively for each of these processes (Table 1; Fig. 5). Inhibitionof these processes caused a measurable drop in the oxygenconsumption rate for both cell types, with the exception of theCa2+-ATPases and xanthine oxidase. The relative demand fromeach of these processes was largely maintained, with proteinsynthesis and DNA and/or RNA synthesis still accounting for the

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majority of mitochondrial respiration in both cell types. However,the absolute oxygen consumption rates that were sensitive toinhibition of protein synthesis, DNA and/or RNA synthesis andNa+/K+-ATPase were significantly higher in hESCs, indicating thatthese processes consume more ATP in hESCs than in NSCs.

We considered the possibility that differences in cell division ratesbetween hESCs and NSCs could account for some of the observeddifferences in ATP turnover. Hence, generation times were determinedthrough time-lapse imaging. Surprisingly, the generation time ofNSCs was significantly shorter than that of hESCs (H9 hESCs at20.3 hours compared with NSCs at 14.2 hours; I6 hESCs at 24.2hours compared with NSCs at 14.1 hours) (Fig. 6A–C; supplementarymaterial Movie 1). No significant differences in cell death rates wereobserved between hESCs and NSCs (Fig. 6D). In conclusion, thehigher ATP turnover rate in hESCs cannot be explained by a highergrowth rate. Instead, processes that consume ATP in the sameproportion as at lower growth rates must be increased; these mightinclude autophagy or secretion of macromolecules.

Electron microscopy showed that there were hallmarks ofmacromolecule secretion in hESCs but not in NSCs (Fig. 7A,B).hESCs typically contained clusters of single-membrane-boundvesicles with homogeneous content and a less electron-dense core(Fig. 7A–D). Although no definite exocytotic events were observed,electron micrographs suggest a goblet-cell-like secretionmechanism (Fig. 7C), which is supported by previous resultsshowing the secretion of cytosolic and cytoskeletal proteins in theproteome of hESC-conditioned medium (LaFramboise et al., 2010).By contrast, evidence of autophagy was uncommon, but both linescontained large multivesicular bodies signifying heterophagy. Toassess the relative rates of protein secretion directly, we analysedmedium conditioned for 10 hours by hESCs and NSCs and founda significantly higher concentration of secreted proteins in mediumconditioned by hESCs (Fig. 8).

Thus, the high ATP production rate in hESCs might be requiredto support RNA synthesis, protein synthesis, ion pumping andcytoskeletal rearrangements during macromolecule secretion by agoblet-cell-like mechanism, in which large parts of the cell,including cytosolic and cytoskeletal components, are secreted.

DiscussionTo our knowledge, this is the first report of the bioenergetics ofhESC differentiation. Our principal finding is that hESCs maintaina high rate of mitochondrial oxidative phosphorylation, whichfunds their high ATP demand for processes other than growth. Wereport a discrepancy wherein decreasing energy turnover is

Fig. 5. The relative division of energy-consuming reactions is largelymaintained between hESCs and NSCs. The relative division of totalrespiration is shown, corresponding to the values in Table 1.

Table 1. Rates of energy-consuming reactions in hESCs and NSCs

Respiration rate±s.e.m. Significance Percentage of(pmols of O2/minute/10 g of cell protein) between total respiration

Process hESCs NSCs respiration rates hESCs NSCs

Protein synthesis 32.7±5.3 (n3) 19.2±1.5 (n6) P0.013 20.0 22.9DNA and/or RNA synthesis 18.8±0.7 (n3) 13.5±1.4 (n4) P0.030 11.5 16.1Proteasome 9.9±2.7 (n3) 8.3±1.4 (n3) NS 6.1 10.0Tubulin turnover 5.0±1.7 (n3) 6.8±1.3 (n3) NS 3.1 8.1Actin turnover 9.4±1.2 (n3) 3.2±1.9 (n4) NS 5.8 3.8Na+/K+-ATPase 12.5±1.1 (n3) 4.3±2.0 (n3) P0.023 7.6 5.1Non-mitochondrial 37.9±1.6 (n18) 20.3±1.1 (n23) P<0.001 23.2 24.3Unidentified 37.2 8 22.8 9.6Sum 163.4 83.6 100 100

The rates of specific ATP consumers were measured as the decrease in respiration rate caused by complete inhibition of the process. The significance betweenthe rates was calculated using a Student’s t-test. The relative division of total respiration is shown and presented graphically in Fig. 5.

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concomitant with increasing cell division rate during the transitionfrom hESCs into NSCs, which might be explained bymacromolecule secretion in hESCs.

ESCs have a fundamentally different in vivo biological rolecompared with that of somatic progenitor cells, which can remain

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quiescent for long periods of time. It seems sensible that these celltypes would be bioenergetically tuned towards their normaldevelopmental roles. Using a novel experimental platform enablingmeasurement of oxygen consumption rates and glycolytic rates innormal adherent cultures, we discovered that hESCs had an elevated

Fig. 6. Cell generation times for hESCs and NSCs. (A)hESCcolony labelled with Hoechst 33342 under time-lapse imaging.Cells undergoing mitosis or cell death are labelled. (B)hESCmean cell generation times deduced from individual coloniesfrom the I6 and H9 hESC lines. (C)Estimated mean (+s.e.m.)cell generation times for I6 and H9 hESCs (n12 each) andNSCs (n4 each). (D)Mean (+s.e.m.) frequency of cell deathover a 24-hour period. Significance between values wascalculated using a Student’s t-test. **P<0.01.

Fig. 7. Secretory-like vesicles are uniquely present inhESCs. (A)Transmission electron micrographs of a typicalfield inside an I6 hESC colony with clusters of secretoryvesicles in most cells. (B)Typical I6 NSC; no structuresresembling the vesicle clusters of hESCs are seen in NSCs.(C,D)Magnifications of hESC secretory-vesicle-rich areasof H9 hESC and I6 hESCs lines, respectively. The arrowmarks the goblet-cell-like secretion mechanism. Panel Dshows that secretory vesicles are often surrounded bymitochondria and granules resembling polysomes. v,secretory vesicles appearing as membrane-bound structureswith homogeneous content; m, mitochondrion; n, nucleus.

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rate of oxidative phosphorylation compared with hESC-derivedNSCs, but without increased glycolysis. The hESC lines weinvestigated also had a relatively high mitochondrial biogenesiscompared with NSCs and HDFs (Fig. 2), and overall our datasupport a model whereby mitochondrial biogenesis is decreasedupon the transition from hESCs into early progenitors but increasedagain on differentiation into more specialised cell types, such asthe neuronal population examined in the present study. The changesin mitochondrial biogenesis correlated with the changingcontribution of oxidative phosphorylation to ATP output duringdifferentiation. The higher maximal respiratory capacity of hESCscould be caused by the higher matrix-to-mitochondria andmitochondria-to-cell-volume ratios and the greater amounts ofrespiratory complexes, as indicated by the abundance of mtDNAand cytochrome c. Other studies have presented data suggestingthat some indicators of mitochondrial content might increase overallduring hESC differentiation in a mixed cell populationdifferentiating in the presence of serum (Armstrong et al., 2010;Saretzki et al., 2008). This could reflect the preferential inductionof other cell types, such as cardiomyocytes, in the presence ofserum, but conclusions are difficult to draw on the basis of whole-cell fluorescent measurements because changes in cell size, andboth mitochondrial and plasma membrane potentials, will affectthese readings (see Fig. 4D). We have shown in the present studythat, in a pure population of hESC-derived NSCs, mitochondrialcontent and activity are reduced compared with those of the parentcells. Further study will be needed to assess the mitochondrial andbioenergetic changes in other cell lineages. However, preliminarydata from a cardiac differentiation model suggest a uniquemitochondrial development for beating compared with non-beatingcells (M.J.B., unpublished), highlighting further the importance ofstudying pure populations of cells.

From these observations, we inferred that hESCs might have agenetic programme that facilitates their high mitochondrial activityand identified expression patterns consistent with this for the‘master regulators’ PGC-1, PGC-1 and RIP140. The PGC-1

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coactivators have been widely studied in other models and shownto be potent regulators of mitochondrial gene expression (Arany etal., 2007; Fritah et al., 2010; Lelliott et al., 2006; Lin et al., 2003;Meirhaeghe et al., 2003; Powelka et al., 2006; Seth et al., 2007).We observed high levels of expression of the mRNA encodingcoactivators PGC-1 and PGC-1 in hESCs and, interestingly,also in the cancer cell line MCF7, pointing to potentialcommonalities in energy metabolism. MCF7 cells have beenreported to obtain 80% of their ATP from oxidative phosphorylation(Guppy et al., 2002). Nearly all the other primary somatic celltypes studied showed reduced expression of the mRNA encodingPGC-1 and PGC-1, together with increased expression of thatencoding the corepressor RIP140. Recent publications supportthese observations by showing that many other genes involved inmitochondrial biogenesis, including those encoding the mtDNAtranscription factor TFAM and the mitochondrial RNA polymerasePOLRMT, are also downregulated upon hESC and iPSCdifferentiation (Armstrong et al., 2010; Prigione et al., 2010).Whether these expression changes occur as a programme ofdifferentiation or whether they change in response to reducedenergy demand remains an unanswered question. Increasing energydemand in fibroblasts through mitochondrial uncoupling has beenreported to induce the PGC-1 coactivators (Rohas et al., 2007).The expression levels of the PGC-1 coactivator could decrease ifenergy demand fell in the early stages of differentiation. However,as we see a strong induction of PGC-1 upon neuronaldifferentiation from NSCs, without any increase in ATP demand,and a return towards reliance on oxidative phosphorylation-generated ATP, a developmental genetic programme might also bea driving force in both of these transitions. Manipulation of PGC-1 or RIP140 in NSCs and HDFs was insufficient to drive achange in the bioenergetics, and so other genes might oppose orlimit their effects in these cells.

Measurements of mitochondrial membrane potential showed thathESCs and NSCs had a similar m of approximately –130 mV. Thehigher rate of oligomycin-sensitive respiration in hESCs in theabsence of a significantly different mitochondrial membrane potentialsuggests a parallel increase in the rate of ATP-supplying and-demanding processes. Concerted regulation of demand and supplycould be in favour of avoiding increases in m and an associatedincrease in the production of reactive oxygen species (Brand, 2010;Murphy, 2009). A breakdown of ATP budgets revealed that in bothhESCs and NSCs ATP was consumed through largely the samepathways. Protein and nucleic acid synthesis together accounted forthe largest portion of the energy budget (hESCs, 41%; NSCs, 52%of mitochondrial respiration), indicating that most of the ATP drivesmacromolecule synthesis. As the total energy budget was greater inhESCs than in NSCs, this indicated a faster macromolecule synthesisrate in hESCs; however, in the absence of increased cell division,these extra macromolecules must ultimately be degraded or secreted.hESCs have been shown to secrete a diverse range of chemokines,cytokines, growth factors and matrix remodelling enzymes, alongwith cytosolic and cytoskeletal components (Bendall et al., 2009;LaFramboise et al., 2010). We found that hESCs had morphologicalindications of goblet-cell-like secretion and secreted significantlymore protein into the extracellular environment than did NSCs.Further study is warranted to investigate the importance of thissecretory activity. Secretion could be a driving force necessitating anincreased rate of biosynthesis; however, an elevated biosynthesisrate in the absence of faster division might also drive secretioninstead of increasing cell size. Goblet-cell-like hESCs have also

Fig. 8. hESCs secrete more protein than NSCs. (A)Proteins precipitatedfrom the medium conditioned by I6 hESCs or NSCs for 10 hours weresubjected to SDS-PAGE and stained with GelCode Coomassie Blue stain.Loading of precipitated material was corrected for the amount of cell proteinin each experiment. Whole-cell lysate lanes are shown for 85g of protein.The control lane is from a medium- or Geltrex-only precipitation. The gels arerepresentative of two independent experiments. (B)Quantification of totalprecipitated protein using densitometry. The intense band at 55 kDa, alsovisible in the control, was excluded from the quantification. Values aremeans+s.d. for three independent experiments. Significance between valueswas calculated using a Student’s t-test. **P<0.01.

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been observed by Sathananthan and colleagues (Sathananthan et al.,2002). hESCs are known to divide relatively slowly – much slowerthan mouse ESCs – so it is plausible that in hESCs the division ratemight not keep pace with the programme driving biosynthesis, butthis hypothesis will require further study. The expression of potentoncoproteins such as c-Myc is a characteristic of ESCs and could bean example of such a programme. Myc itself is an important regulatorof ribosome biogenesis and protein synthesis, and also a regulator ofmitochondrial activity and biogenesis (Fan et al., 2010; Kim et al.,2008; Li et al., 2005; van Riggelen et al., 2010). The importance ofbiosynthesis and energy metabolism in ESCs has also beenhighlighted in recent studies (Dejosez et al., 2010; Wang et al.,2009).

In summary, we provide what we believe to be the first evidencethat the energy demand for many cell processes in hESCs iselevated relative to their division rate and funded primarily by ATPgenerated through mitochondrial oxidative phosphorylation.Developing embryos of several species studied in vitro show alarge increase in oxygen consumption at the blastocyst stage,during which protein synthesis is at its maximal rate, and it hasbeen estimated that the majority of ATP comes from oxidativephosphorylation during this phase (Houghton et al., 1996; Sturmeyand Leese, 2003; Thompson et al., 1996). In this respect, hESCsmight mirror the metabolic characteristics of cells of the developingblastocyst during this important phase of expansion.

Materials and MethodsCell culture and differentiationhESC lines I6, H9 and BG01 were maintained as described previously (Zeng et al.,2004) or were grown on Geltrex-coated dishes (1:50) (Invitrogen, Carlsbad, CA) inthe same medium previously conditioned for 24 hours on inactivated mouseembryonic fibroblasts (MEFs). B6 mouse ESCs were grown in the sameunconditioned medium but supplemented with 1000 units/ml leukaemia inhibitoryfactor (LIF) (Millipore) instead of fibroblast growth factor 2 (FGF2). For hESCdifferentiation, colonies were detached with collagenase IV (1 mg/ml), triturated andsuspended as embryoid bodies for up to 16 days or re-plated on Geltrex-coateddishes for up to 12 days, both in standard ES medium without FGF2. mESCs weredifferentiated as embryoid bodies in standard ES medium without LIF.

NSC derivation, culture and differentiation were performed as described previously(Swistowska et al., 2010; Swistowski et al., 2009). H9 hESC-derived NSCs werefrom Millipore. BJ fibroblasts were from the ATCC and grown in high-glucoseDulbecco’s modified Eagle’s medium (DMEM) containing sodium pyruvate and L-glutamine (Invitrogen) plus 10% foetal bovine serum (FBS) and penicillin andstreptomycin. All other cell types used for RNA expression analysis were fromInvitrogen and cultured briefly using the medium recommended by the manufacturerbefore RNA isolation (for details, see supplementary material Table S1).

To calculate the cell generation times, NSCs were imaged at 24 hours afterseeding, using phase-contrast microscopy in a humidified chamber with 5% CO2, at4-minute intervals for 8 hours. At 24 hours after seeding, hESCs were labelled with2 g/ml Hoechst 33342 (Molecular Probes H3570) and fluorescent images wererecorded at 8-minute intervals with minimal excitation intensity for 24 hours. Cellswere randomly selected from several fields, tracked by eye and division or deathevents were recorded. The mean generation time and frequency of death werecalculated.

Analysis of conditioned mediaFor the analysis of conditioned media, I6 hESCs or NSCs were grown to highdensity on Geltrex-coated 6-cm-diameter dishes in normal growth medium as above,rinsed twice and then incubated in DMEM with Ham’s F12 plus Glutamax for 10hours. After collection, the medium was centrifuged to remove debris, and theprotein component was precipitated using a 1:8:1 ratio of medium, acetone andtrichloroacetic acid, respectively, at –80°C for 1 hour. Precipitates were centrifugedat 18,000 g for 15 minutes at 4°C and resuspended in a volume of SDS-PAGEloading buffer proportional to the relative amounts of cellular protein harvested fromthe dish. Total cell lysates were made from the monolayers by using mammalianprotein extraction reagent (Pierce) containing protease inhibitor cocktail (Roche).The cell lysate protein levels were quantified according to the Biuret method andconfirmed on test gels with GelCode Coomassie Brilliant Blue staining (ThermoScientific). Precipitated samples were subjected to SDS-PAGE alongside the proteinlysates and stained as above. Gels were scanned and lane intensities were quantifiedusing densitometry in ImageJ software (NIH).

Measurement of respiration and acidification rates using a Seahorse XF-24analyserRespiration and acidification rates were measured on adherent cells using a SeahorseXF-24 analyser (Seahorse Bioscience, North Billerica, MA). For hESCs and NSCs,the XF24 V7 assay plates were coated with Geltrex (1:50). Cells were seeded atapproximately 24 hours before measurement in their normal growth medium; NSCswere seeded at 7�104 cells per well and hESCs as colonies totalling ~3�104 cellsper well. The assay was performed in bicarbonate-free DMEM (Sigma D-5030)supplemented with 15 mM glucose, 2 mM L-glutamine, 0.5 mM sodium pyruvateand 0.4% BSA. Cells were washed twice and preincubated in this medium for 1 hourbefore measurement. Oligomycin was used at 0.2 g/ml; carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was titrated in four injections to 1.5 Mfor hESCs, NSCs and D-35s, and 2 M for HDFs; and rotenone and antimycin Awere added at 1 M and 2 M, respectively. Oxygen consumption values werecalculated from 3-minute measurement cycles by the XFReader software Version 1.4updated with a recent correction (Gerencser et al., 2009). Acidification rates weretaken as the mean rate from the second and third baseline readings. After the assay,a standard protein assay was performed. Alternatively, with the D-35 cells, somewells were fixed with 4% paraformaldehyde for immunocytochemical staining, asdescribed below.

ATP demand assayATP demand was measured using the Seahorse XF-24 analyser as above with theaddition of 10 mM HEPES to the assay medium. The basal respiration rate wasrecorded and then the selective inhibitors were injected and respiration recorded forthe following 2–5 minutes. Small changes from vehicle-only controls, which wereincluded on each plate, were subtracted to yield the specific effects of the inhibitors.The inhibitors used for each cellular process, and the concentrations tested, include:protein synthesis (cycloheximide, 0.01–50 g/ml); DNA and/or RNA synthesis(actinomycin D, 0.1–10 M); proteasome (MG132, 0.1–50 M); tubulin dynamics(nocodazole, 0.1–50 M); actin dynamics (latrunculin A, 0.1–3 M); Na+/K+-ATPase(ouabain, 1 mM); sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)(thapsigargin, 1–1000 nM); plasma-membrane Ca2+-ATPase (PMCA) (LaCl3, 1–4mM); and xanthine oxidase (allopurinol, 10–1000 M). Mitochondrial respirationwas subsequently fully inhibited with rotenone and antimycin A, as described above.All compounds were from Sigma, except MG132 (Calbiochem).

Wide-field imaging for determination of plasma and mitochondrialmembrane potentialsImaging was conducted in eight-well LabTek I (Nunc, Rochester, NY) coverglasschambers. For hESCs, the glass was coated with 0.1% gelatin and the colonies weregrown on a feeder layer of MEFs. For NSCs the glass was coated with poly-ornithine(20 g/ml) and then Geltrex (1:100). Tetramethylrhodamine methyl ester (TMRM)(7.5 nM; Invitrogen), plasma membrane potential indicator (PMPI) (1:200; no.R8042 FLIPR membrane potential assay kit from Molecular Devices; Sunnyvale,CA) and Na-tetraphenylborate (1 M) were continuously present in the experimentalbuffer (120 mM NaCl, 1.75 mM KCl, 0.2 mM KH2PO4, 10 mM TES, 2.5 mMNaHCO3, 0.6 mM Na2SO4, 1.3 mM CaCl2, 2 mM MgCl2, 15 mM glucose, 2 mML-glutamine and 0.5 mM Na-pyruvate, pH 7.4) at 37°C. Imaging was performed ona Nikon Eclipse Ti-PFS inverted fluorescence microscope controlled by NIS Elements3.1 (Nikon, Melville, NY) and equipped with a S-Fluor 20�/0.75 NA lens, aLambda LB-LS17 light source (Sutter Instruments, Novato, CA) a Cascade 512Bcamera (Photometrics, Tucson, AZ) and a MS-2000 motorised stage (ASI; Eugene,OR). The filter sets given as excitation–dichroic mirror–emission in nm were: forPMPI 500/24–520LP (long pass)–542/27 and for TMRM: 582–593LP (all fromSemrock, Rochester, NY)–610LP (Omega Optical, Brattleboro, VT). Images weretaken at 10-second intervals, typically cycling in the 2–12 stage positions. Toestablish the K+ equilibrium potential, a cocktail of drugs (KEC) was added includingK+-channel activators: flupirtine (10 M), NS309 (10 M) and diazoxide (100 M);chloride channel co-transport inhibitors: R-(+)-IAA94 (100 M), R-(+)-DIOA (10M), bumetanide (80 M); and the sodium channel inhibitor tetrodotoxin (1 M).For calculation of m mitochondria were completely depolarised with a cocktail(MDC) of valinomycin (1 M), oligomycin (2 M), FCCP (1 M) and myxothiazol(1 M). Complete cell depolarisation was induced with a cocktail (CDC) ofionophores: gramicidin (10 M), nigericin (10 M), monensin (10 M) and theglycolysis inhibitor iodoacetate (500 M). This cocktail also included the componentsof the MDC. Calculation of p and m from the measured fluorescence intensitieswere performed as described in the supplementary methods.

Confocal imaging of mitochondrial volume ratiosCells were grown in LabTek I chambers as described above and were loaded withcalcein-AM (1 M; Invitrogen) and MitoTracker Red (hESC, 50 nM; NSC, D-35and HDF, 25 nM; Invitrogen) in the above experimental buffer for 20 minutes, thenimaged in experimental buffer with a Zeiss LSM 510 laser scanning confocalmicroscope. Single planes of 1024�1024 pixel images at 44 nm/pixel resolutionwere recorded using a Plan-Apochromat 100�/1.4 NA oil lens. Calcein andMitoTracker Red were excited simultaneously using the argon 488 nm and helium–neon 543 nm laser lines at high intensity, and fluorescence emissions were collectedin the range of 500–530 nm and above 560 nm, respectively. To record serial optical

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sections without recording photo-damaged areas, random images were acquired atdifferent x- and y-locations, and the z-plane was advanced from the bottom of theculture to the highest focal plane in steps of 1–1.5 m. Images were binarised byhighpass filtering and locally adaptive thresholding using Image Analyst MKIIsoftware (Image Analyst Software, Novato, CA). To calculate mitochondria-to-cell-volume ratios, the total number of mitochondrial pixels was divided by the totalnumber of cytosolic pixels in the complete image set and multiplied by 0.67 – astereologic correction factor (Weibel and Paumgartner, 1978) that accounts for theprojection and clipping of mitochondria by the optical section. See the supplementarymethods.

Transmission electron microscopySample preparation and processing are described in the supplementary methods. Fordetermination of matrix volume ratios, mitochondria were selected in a uniformrandom approach and imaged at �105,000 magnification. Matrix-to-whole-mitochondrial-volume ratios were calculated by the Cavaileri estimator using Stereo-Investigator (MBF Bioscience; Williston, VT).

mtDNA copy number assessmentAbsolute mtDNA copy number was assessed by comparing PCR amplification of amitochondrial amplicon [human, NADH-ubiquinone oxidoreductase chain 1 (ND1);mouse, cytochrome b] with a nuclear amplicon (human, - and/or g-globin; mouse,glucagon) from DNA isolated using a Qiagen DNA mini kit. Primer sequences arein supplementary material Tables S2 and S3. DNA templates were made of regionsspanning each amplicon by PCR amplification. Dilutions from 1�108 down to1�104 copies of the isolated DNA were used to generate a standard curve forquantification.

Western blots and immunocytochemistryTotal cell lysates were made using the mammalian protein extraction reagent (Pierce)containing protease inhibitor cocktail (Roche). Gels were run and proteins transferredusing standard techniques. Antibodies were: anti-cytochrome c (no. 556433, BDPharmingen); anti-VDAC (no. MSA03, MitoSciences); anti-MnSOD (no. sc-30080,Santa Cruz Biotechnology); anti-CuZnSOD (no. sc-11407, Santa CruzBiotechnology). Immunocytochemistry and staining procedures were performed asdescribed previously (Zeng et al., 2003).

RNA isolation and cDNA synthesis for real-time PCRRNA was isolated using TRIzol (Invitrogen) and was treated with DNase-I (Sigma).cDNA was synthesised using the Superscript III reverse transcriptase system(Invitrogen). Real-time PCR was performed using the PerfeCTa SYBR Green 2�mix (Quanta Bioscience) on an Applied Biosystems 7900HT fast real-time PCRmachine. Primer details and sequences are presented in supplementary materialTables S4 and S5.

Lentiviral transductioncDNA encoding PGC-1 was obtained from Open Biosystems (clone ID: 40146993)and cloned into a pLenti CMV/TO Neo vector (Addgene: 17292). The shRNAtargeting RIP140 was constructed using sense: 5�-gcagcagtattctcgagaaGT -GTGCTGTCCttctcgagaatactgctgc-3� and antisense 5�-gcagcagtattctcgagaa -GGACAGCACACttctcgagaatactgctgc-3� sequences and expressed in a pLenti X2Puro vector (Addgene number 17296). The uppercase nucleotides indicate the targetsequence. The empty pLenti CMV/TO Neo vector or an shRNA against luciferase(Addgene: 21472) were used as controls. This lentiviral system has been describedpreviously (Campeau et al., 2009). All vector inserts were sequenced to confirmidentity. For packaging lentiviruses, human embryonic kidney HEK-293FT cellswere used (Invitrogen). Lentiviral supernatants were concentrated usingultracentrifugation and titers were used to infect ~80–90% of the cells, as judged bya control virus with a cytomegalovirus promoter driving the expression of greenfluorescent protein (GFP). Cells were selected with neomycin or puromycin for 1week before analysis. Two separate derivations were made for I6 NSCs and HDFsfor bioenergetic analyses.

This work was supported by the California Institute for RegenerativeMedicine Grant CL1-00501-1 (X.Z.) and the Larry L. HillblomFoundation (X.Z.). RS1-00163-1 (D.T.M., laboratory of Dale E.Bredesen), National Institutes of Health grants PL1 AG032118 (A.L.O.,A.A.G., C.V. and M.D.B.), P01 AG025901, P30 AG025708 and R01AG033542 (M.D.B.), the W. M. Keck Foundation (A.A.G. andM.D.B.), and The Ellison Medical Foundation (AG-SS-2288-09)(A.L.O. and M.D.B.). We thank Linzhao Cheng at the Johns HopkinsUniversity for providing the iPSC line. We thank Eric Campeau at theUniversity of Massachusetts for providing the lentiviral vectors.Deposited in PMC for release after 12 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/3/348/DC1

Supplementary methods available online athttp://www.buckinstitute.org/sites/default/files/Birket_et_al_Supplementary_material.pdf

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