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doi: 10.1111/joim.12247

Metabolic regulation of stem cell function R. J. Burgess*, M. Agathocleous* & S. J. Morrison From the Department of Pediatrics, Howard Hughes Medical Institute, Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

Abstract. Burgess RJ, Agathocleous M, Morrison SJ (Howard Hughes Medical Institute, Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA). Metabolic regulation of stem cell function (Key Symposium). J Intern Med 2014; 276: 12–24. Stem cell function is regulated by intrinsic mechanisms, such as transcriptional and epigenetic regulators, as well as extrinsic mechanisms, such as short-range signals from the niche and longrange humoral signals. Interactions between these regulatory mechanisms and cellular metabolism are just beginning to be identified. In multiple systems, differentiation is accompanied by changes in glycolysis, oxidative phosphorylation and the levels of reactive oxygen species. Indeed,

Introduction The development and maintenance of healthy tissues depends upon appropriate stem cell regulation. During embryonic development, pluripotent embryonic stem (ES) cells divide rapidly to form the blastocyst and then give rise to multipotent somatic stem cells during organogenesis. Multiple distinct types of somatic stem cells arise during foetal development to form and grow tissues (Fig. 1). Some of these stem cell populations persist postnatally to regenerate adult tissues. For example, haematopoietic stem cells (HSCs), forebrain neural stem cells and intestinal epithelial stem cells are required throughout life to replenish cells that turnover in their respective tissues. Although stem cells persist throughout life in many tissues, their properties shift over time to match the changing growth and regeneration demands of the tissues [1, 2]. Somatic stem cells divide rapidly during foetal development to support organogenesis but are often quiescent in adult tissues, only entering the cell cycle periodically to maintain tissue homoeostasis.

*These authors are contributed equally.

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metabolic pathways regulate proliferation and differentiation by regulating energy production and the generation of substrates for biosynthetic pathways. Some metabolic pathways appear to function differently in stem cells as compared with restricted progenitors and differentiated cells. They also appear to influence stem cell function by regulating signal transduction, epigenetic marks and oxidative stress. Studies to date illustrate the importance of metabolism in the regulation of stem cell function and suggest complex cross-regulation likely exists between metabolism and other stem cell regulatory mechanisms. Keywords: differentiation, metabolism, self-renewal, stem cell.

Stem cells are often regulated differently than restricted progenitors in the same tissue. For example, HSCs depend upon key transcription factors [3–5], epigenetic regulators [6–9], signal transduction mechanisms [10, 11] and microenvironments [12, 13] that are not required by most restricted haematopoietic progenitors. Whether metabolism regulates these stem cell mechanisms or these mechanisms regulate metabolism, the abundance of mechanisms that distinguish stem cells from restricted progenitors suggests that stem cells also differ metabolically from restricted progenitors. However, the data remain limited because many aspects of cellular physiology remain unstudied in somatic stem cells. Stem cells harbour a unique combination of properties, including the maintenance of an undifferentiated state, self-renewal potential and often multipotency. Stem cells reversibly enter and exit the cell cycle over time, giving rise to more stem cells (self-renewal) as well as differentiated progeny. A fundamental question is whether these properties depend upon stem cell-specific metabolic adaptations. This question has only been investigated to a limited extent, and the data are often undermined by technical impediments, such as the need to perform experiments in culture or

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Key Symposium: Stem cell metabolism

In vitro Ectoderm

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Fig. 1 There are many different kinds of stem cells that exist at different times during development and in different tissues. Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst. Induced pluripotent stem (iPS) cells have properties that are very similar to ES cells, but arise from the reprogramming of more mature somatic cells [124–126]. ES cells and iPS cells are studied almost exclusively in culture. During organogenesis, pluripotent cells of the early embryo give rise to more specialized somatic stem cells that often remain multipotent but become specialized to individual tissues. For example, haematopoietic stem cells (HSCs) give rise to many different kinds of blood and immune system cells but not to cells in other tissues. In contrast, neural stem cells are regionally specialized to form only cell types that arise in a particular region of the nervous system. There are many kinds of somatic stem cells in different tissues. Stem cells persist throughout life in a number of tissues, including the haematopoietic system, the forebrain and various epithelia. Stem cells in adult tissues typically remain multipotent but tissue specialized, although they have different properties than foetal stem cells in the same tissues [1]. In addition to the stem cells shown for illustration purposes in the figure, there are many other kinds of somatic stem cells, such as in muscle, skin and breast epithelium. SC, stem cell; AGM, aorta–gonad–mesonephros region; CNS, central nervous system.

the use of heterogeneous progenitor populations rather than highly purified stem cells. Much of the data must, therefore, be interpreted cautiously because somatic stem cell properties can be profoundly changed in culture [14], and heterogeneous progenitor populations have uncertain similarity to the rare stem cells they contain.

For almost a hundred years, it has been appreciated that metabolic changes occur in concert with developmental changes during embryogenesis [15], suggesting that metabolism could potentially regulate fate determination. Systemic and local cues reflecting nutritional status influence stem cell function in multiple tissues [16–19], and emerging ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 12–24

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Key Symposium: Stem cell metabolism

evidence supports the idea that there are metabolic differences between stem cells and other cells (e.g. [20–25]). In this review, we integrate this emerging understanding of stem cell metabolism with knowledge of other stem cell regulatory mechanisms and discuss the implications for future research. Pluripotent stem cells are particularly dependent upon glycolysis Given the energy and biosynthetic demands of cell division, metabolism would be expected to differ between dividing and nondividing cells. Glucose is metabolized by glycolysis to pyruvate, which either undergoes reduction by lactate dehydrogenase to lactate or enters the mitochondria to be decarboxylated by pyruvate dehydrogenase to acetyl-CoA for use in the tricarboxylic acid (TCA) cycle (Fig. 2). The conversion of pyruvate to lactate or acetyl-CoA is determined, in part, by oxygen availability. Under hypoxic conditions, acetyl-CoA production is curtailed and pyruvate is converted to lactate instead [26]. Another variable is the rate of cellular growth or proliferation. Growth requires the production of macromolecules and ATP. In some

cancer cells, this may be accomplished through the Warburg effect. The Warburg effect is characterized by increased glycolysis despite normoxia, with some pyruvate being converted to lactate rather than entering the TCA cycle [26]. This allows products of glycolysis to be diverted to anabolic biosynthetic pathways rather than undergoing complete oxidation to carbon dioxide and water in the TCA cycle. Whilst cellular growth and proliferation can alter metabolism as a consequence of the need to generate substrates for biosynthetic pathways, the implications for many stem cell populations are uncertain. For example, most adult stem cell populations are quiescent most of the time, which calls into question the extent to which they need to divert products of glycolysis for anabolic growth. In contrast to adult stem cells, ES cells divide rapidly, with an unusually short G1 phase [27]. Therefore, they might be expected to be particularly dependent upon glycolysis to support cellular growth and division. Consistent with this idea, ES cells have a high rate of glycolytic lactate produc-

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Threonine

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Fig. 2 Metabolic pathways discussed in this review. Glucose is metabolized through glycolysis to generate metabolites that serve as substrates in multiple biosynthetic pathways [26]. Pyruvate can enter the mitochondria to be metabolized to acetyl-CoA for entry into the tricarboxylic acid (TCA) cycle or it can be converted to lactate in the cytoplasm. Metabolites are shown in black, metabolic pathways in red and enzymes in green. Not all metabolic intermediates, reaction cofactors and pathways are shown.

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tion compared with differentiated cardiomyocytes [28], although they do still exhibit oxidative phosphorylation [29, 30]. Differentiation of ES cells to neural stem cells is accompanied by increased glycolysis and decreased oxidative phosphorylation, although glycolysis declines upon terminal differentiation to neurons [30]. Reprogramming of fibroblasts to induced pluripotent stem (iPS) cells is associated with increased glycolysis prior to the appearance of pluripotency markers [31]. These studies suggest that pluripotent stem cells, and perhaps multipotent stem cells, have a higher rate of glycolysis than more differentiated cells. Much of the evidence for increased glycolysis in stem cells is based on the observation that these cells produce more lactate. Lactate production and the siphoning of glycolytic products for biosynthetic pathways are not incompatible despite the fact that each consumes carbon. Lactate production allows glycolysis to be accelerated because the conversion of pyruvate to lactate provides the nicotinamide adenine dinucleotide (NAD+) necessary for glycolysis [26]. Multiple glycolytic intermediates upstream of pyruvate can be siphoned off to biosynthetic pathways (Fig. 2). 3-Phosphoglycerate can be used to make serine and glycine, which are needed in amino acid, lipid and nucleotide biosynthesis. Dihydroxyacetone phosphate can be used to make phospholipids. Glucose-6-phosphate can be diverted to the pentose phosphate pathway [26]. Glycolysis in pluripotent stem cells is functionally important for maintaining the pluripotent state. Inhibition of glycolysis in human ES cells results in cell cycle arrest and apoptosis, in line with the idea that glycolysis is used by rapidly dividing cells with increased anabolic demands [29, 32]. Inhibition of glycolysis also prevents reprogramming of fibroblasts to iPS cells, without affecting fibroblast proliferation [31, 33]. Whilst glycolysis may be important at some level in all cells, these data suggest it is particularly important for the maintenance of pluripotent stem cells. The apparently high rate of glycolysis in pluripotent stem cells could reflect an inability of their mitochondria to fully support oxidative phosphorylation. Some researchers have proposed that the mitochondria in mouse [28] and human [34] ES cells, as well as human iPS cells [35], are immature compared to those in more differentiated cells, with lower transcription of electron transport chain components. Mouse epiblast stem cells, which are

Key Symposium: Stem cell metabolism

pluripotent cells collected at a later stage of embryonic development than ES cells, contain more morphologically mature mitochondria than mouse ES cells but are obligatorily glycolytic due to low cytochrome C expression [29]. Moreover, human ES cells have a similar mitochondrial mass and oxygen consumption rate as fibroblasts when normalized to protein content, but their mitochondria consume rather than produce ATP [32]. Nonetheless, mitochondrial function does contribute to the maintenance of pluripotent cells. Uncoupling protein 2 (UCP2), a mitochondrial inner membrane protein more highly expressed in pluripotent stem cells than in fibroblasts, may shunt pyruvate away from the mitochondria and stimulate glycolysis [32]. UCP2 overexpression impairs human ES cell differentiation, although UCP2 knockdown does not promote differentiation [32]. Whilst pluripotent stem cells have high levels of glycolysis, oxidative phosphorylation remains important. Amino acids have metabolic functions beyond protein synthesis, and mouse ES cells are particularly dependent upon threonine [25]. Withdrawal of individual amino acids from ES cell cultures revealed that only threonine is required at high extracellular concentrations [25]. Threonine dehydrogenase, the enzyme that initiates threonine catabolism, is highly expressed in mouse ES cells, and its loss [36] or threonine withdrawal from the medium [25] causes ES cell growth arrest, differentiation and cell death [24]. Metabolic flux analysis with labelled threonine indicates that threonine contributes to the synthesis of glycine, glutamate and acetyl-CoA-derived TCA intermediates [24]. Threonine contributions to glycine ultimately generate acetyl-CoA, 5-methyltetrahydrofolate and Sadenosylmethionine (SAM). Methylation reactions that use SAM convert it to S-adenosylhomocysteine (SAH), and a high ratio of SAM to SAH favours substrate methylation. Reduced threonine dehydrogenase function or threonine restriction decreases intracellular glycine, the SAM to SAH ratio and TCA cycle intermediates [24]. This, in turn, decreases histone methylation, particularly di- and trimethylation of histone H3 lysine 4, an epigenetic mark associated with gene expression [24]. Thus, threonine provides fuel for ES cell divisions and contributes to epigenetic regulation. Increased L-proline concentrations in culture induce the proliferation of ES cells but not various differentiated cells [37]. Blocking L-proline uptake or inhibiting proline catabolism blocks the ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 12–24

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proline-induced morphological changes in ES cells [37]. Under different culture conditions, L-proline can also promote ES cell differentiation [37, 38], motility in culture [39] and teratoma formation when these cells are injected in vivo [37]. The increased motility with proline addition correlates with a global increase in trimethylation of histone H3 lysine 9 and dimethylation of histone H3 lysine 36 [39]. Treatment with vitamin C, which promotes the activity of some histone lysine demethylases [40], reverses proline-induced histone methylation and inhibits proline-induced motility [39]. These results suggest that proline can influence chromatin structure and gene expression, although unlike those for threonine, the metabolic pathways that link proline to histone methylation in stem cells remain unclear. Flux analysis can quantitatively follow the fates of labelled carbon atoms from glucose, making it possible to analyse flux through metabolic pathways within the cell [41]. Given the complexity of the flow of carbon through these pathways and the likelihood of cell type- and context-dependent differences in how the pathways are used, it is important to note that with a few exceptions [25, 42, 43], very little flux analysis has been performed in stem cells. Thus, surprises remain possible regarding the metabolic pathways that are active in stem cells and the differences relative to nonstem cells. Somatic stem cells also appear to depend upon glycolysis The idea that rapidly dividing stem cells are more dependent upon glycolysis than differentiated cells is supported by studies of embryonic progenitors in vivo. Undifferentiated Xenopus embryonic retinal progenitors divide rapidly. They have low oxygen consumption and can generate ATP by glycolysis, and they shift to oxidative phosphorylation upon differentiation to neurons [44]. The balance between glycolysis and oxidative phosphorylation in the cells is intrinsically controlled by differentiation state rather than by environmental oxygen levels, and inhibition of glycolysis impairs cell survival [44]. Differentiation thus appears to induce metabolic changes. Some adult stem cells have also been reported to be glycolytic in vivo. HSCs are thought to be glycolytic because they consume less oxygen, have lower mitochondrial potential [45] and exhibit increased levels of glycolytic metabolites and enzymes in comparison with restricted progenitors or differen16

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Key Symposium: Stem cell metabolism

tiated cells [46]. Glycolysis is regulated by the activity of pyruvate dehydrogenase kinase (PDK) enzymes, which inactivate pyruvate dehydrogenase, therefore promoting glycolytic production of lactate from pyruvate rather than allowing pyruvate to enter the TCA cycle (Fig. 2). Combined deletion of Pdk2 and Pdk4 results in mild defects in HSC reconstituting ability and an increase in proliferation, suggesting that persistent pyruvate dehydrogenase activation impairs HSC function [46]. Nonetheless, it is not clear precisely what metabolic consequences arise from deleting these PDKs in HSCs or how the deletion affects glycolysis and the TCA cycle. Some researchers have attributed the glycolytic metabolism of somatic stem cells to limited oxygen availability in their environment. Multiple studies have demonstrated that the bone marrow, where HSCs reside, is relatively hypoxic [47, 48], including the perisinusoidal microenvironments where most HSCs are found [49, 50]. Hypoxia activates glycolysis by stabilizing the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1a is critical for the switch from oxidative phosphorylation to glycolysis during hypoxia, which maintains ATP production and prevents generation of excessive reactive oxygen species (ROS) [51]. HIF-1a expression is higher in HSCs compared with differentiated cells [45, 52]. Both increased and decreased HIF-1a activities compromise HSC function, although the deficits are modest [52]. Studies in human haematopoietic stem and progenitor cells also support a role for HIF-2a, reduced expression of which increases ROS levels, apoptosis and endoplasmic reticulum stress [53]. Neural stem cells in the dentate gyrus of the hippocampus are thought to reside in a hypoxic environment, given their staining with the hypoxia marker pimonidazole and poor vascularization [54]. Deletion of HIF-1a in these cells reduces Wnt signalling, depletes neural progenitors and reduces neurogenesis [54]. Thus, hypoxia and HIF signalling regulate neural stem cell function, although it remains unclear to what extent this is mediated by changes in energy metabolism. Hypoxia-inducible factor-1a and HIF-2a are hydroxylated by prolyl hydroxylase domain (PHD) enzymes, which promote the interaction of HIF with von Hippel–Lindau protein, leading to HIF ubiquitination and proteasomal degradation [51]. In addition, the PHD protein, factor-inhibiting

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HIF1, inhibits HIF-1a activation by disrupting the interaction of HIF-1a with its co-activator [51]. PHD proteins are members of the dioxygenase family of proteins, which require molecular oxygen and a-ketoglutarate, a TCA cycle intermediate, for their activity. In addition, the PHD enzymes regulating HIF factor stability are negatively regulated by succinate and fumarate in vitro [55]. Therefore, HIF levels in stem cells may be sensitive to both the hypoxic environment and TCA cycle metabolites. Mitochondrial metabolism is also important for HSCs. Conditional deletion of PTPMT1, a mitochondrial phosphatase, from haematopoietic cells results in a striking accumulation of HSCs and severe anaemia caused by a block in differentiation [22]. This is accompanied by decreased oxygen consumption by haematopoietic progenitors, suggesting impaired mitochondrial respiration. PTPMT1 may promote the differentiation of HSCs or their progeny by dephosphorylation of phosphoinositols in the mitochondrial membrane, although the mechanisms that connect PTPMT1, oxidative metabolism and HSC differentiation remain unclear. Reactive oxygen species regulate stem cells Stem cells appear to be particularly sensitive to ROS, which are generated partly through incomplete reduction of oxygen in the electron transport chain during mitochondrial oxidative phosphorylation [56]. ROS react with DNA, proteins and other macromolecules, leading to an accumulation of mutations and misfolded proteins [57]. Increased ROS levels are associated with stem cell depletion and functional defects in several tissues. Forkhead O (FoxO) transcription factors protect against ROS by activating the expression of antioxidant enzymes that detoxify ROS (such as manganese superoxide dismutase and catalase) and DNA repair enzymes that repair ROS-induced damage [58, 59]. Conditional deletion of FoxO1, FoxO3 and FoxO4 from haematopoietic cells in mice increases ROS levels in HSCs and depletes the HSCs [21]. The HSCs appear to be more profoundly impaired by FoxO1/FoxO3/FoxO4 deficiency than restricted myeloid progenitors, which normally have higher levels of ROS than HSCs. The HSC defects in the mice are ameliorated by treatment with the antioxidant N-acetyl-L-cysteine [21]. Amongst FoxO factors, FoxO3 appears to be particularly important because deletion of this family member alone leads to HSC defects [60, 61].

Key Symposium: Stem cell metabolism

Haematopoietic stem cells are not alone in their dependence upon FoxO proteins, as shown by FoxO1/FoxO3/FoxO4 deficiency or FoxO3 conditional deletion in the nervous system leading to neural stem cell defects, including increased ROS levels and stem cell depletion [62, 63]. These defects are only partially rescued by treatment with N-acetyl-L-cysteine and therefore may reflect other FoxO3 functions [63], including transcriptional regulation of differentiation and metabolism [62]. Flux studies in vitro showed that neural progenitors lacking FoxO3 synthesize less glutathione from glutamine and less NADPH from glucose via the pentose phosphate pathway [43]. FoxO transcription factors promote stem cell function through multiple mechanisms. Beyond regulating the enzymes and antioxidants that detoxify ROS, cells have robust mechanisms to maintain redox homoeostasis. We have glimpses of these mechanisms, but our understanding of how they work is limited. Stem cells in multiple tissues depend on the Prdm16 transcription factor for their maintenance [3, 64]. Prdm16 appears to regulate either the generation or the response to ROS because ROS levels are increased in Prdm16deficient neural stem cells, and some neurodevelopmental defects (but not HSC defects) are rescued by N-acetyl-L-cysteine administration [3]. Deletion of the checkpoint kinase ATM also leads to stem cell defects in multiple tissues, including the haematopoietic and nervous systems, causing progressive bone marrow failure in older mice and profound defects in HSC reconstituting ability [65]. ROS levels are increased in Atm-deficient HSCs, and antioxidant treatment partially rescues the HSC defects [65]. Atm deficiency also causes defects in neural stem cell function [66], partly by increasing ROS production and MAPK pathway signalling [67]. It is not clear exactly how Prdm16 or ATM regulates ROS levels. Although increased ROS levels have deleterious effects on stem cells, physiological ROS levels are required for stem cell function. Apart from oxidative phosphorylation, ROS are generated extramitochondrially by membrane-bound NADPH oxidase, which reduces oxygen to superoxide. Mice with genetic disruption of NADPH oxidase 2 have modest neurogenesis defects, and neural stem/ progenitor cells from these mice have reduced neurosphere-forming ability, which is partially rescued by addition of hydrogen peroxide to the culture [68]. Deletion of the mitochondrial ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 12–24

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transcription factor TFAM in the skin of mice prevents transcription of electron transport chain subunits, inhibiting oxidative phosphorylation and ROS generation. This increases the proliferation of epidermal keratinocytes and impairs differentiation, effects that are partially rescued by adding hydrogen peroxide to the culture [69]. Foetal liver HSCs from Akt1 / Akt2 / mice exhibit increased quiescence, decreased reconstituting capacity upon serial transplantation and low ROS levels. Pharmacological increase in ROS, whilst toxic to control cells, improves the proliferation and differentiation of Akt1 / Akt2 / HSCs in culture [70]. Increased ROS levels are required for induction of Wnt signalling and efficient regeneration following amputation of the Xenopus tadpole tail [71]. ROS production is necessary for human mesenchymal stem cell differentiation to adipocytes, and treatment with antioxidants inhibits adipocyte differentiation [72]. In Drosophila haematopoietic progenitors, experimentally increasing ROS levels promotes differentiation, and reducing ROS levels prevents differentiation [73]. Reactive oxygen species thus have multiple roles in regulating stem cell function, although the consequences of changes in ROS levels are cell type specific and depend on the magnitude of the change. It is also important to bear in mind that current tools for measuring ROS are limited and may be insensitive to certain kinds of ROS, very transient bursts of ROS or ROS that are restricted to certain subcellular locations. New techniques may reveal additional facets of ROS regulation and function in cells. For example, genetically encoded redox-sensitive fluorescent probes are being developed that can measure ROS in living cells in vivo [74]. These probes could be particularly useful for studying ROS in somatic stem cells. Reactive aldehydes are another by-product of metabolism that damages cells. Some stem cells exhibit high levels of aldehyde dehydrogenase activity [75], although this is not true of all stem cells, and the aldehyde dehydrogenase activity is not attributable to Aldh1a1 expression, as commonly asserted [76]. Nonetheless, aldehyde dehydrogenases do protect against DNA damage by detoxifying endogenous and exogenous aldehydes [77, 78]. Genetic inactivation of Aldh2 in mice, combined with loss of the Fanconi anaemia DNA repair pathway, results in a catastrophic loss of HSCs and development of leukaemia [77, 78]. This suggests that endogenous aldehydes can cause 18

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Key Symposium: Stem cell metabolism

DNA damage and that a two-tier protective system operates in stem cells to metabolize aldehydes and to repair DNA damage. Aldehyde dehydrogenases are also required for the detoxification of exogenous aldehydes, such as those that arise from ethanol metabolism [77, 78]. These findings have public health implications because acetaldehyde, the reactive aldehyde produced by metabolism of ethanol, may accumulate and cause DNA damage in humans with lower ALDH2 activity (as is observed in some Asians), potentially contributing to increased frequencies of certain cancers. Metabolic regulation of chromatin-modifying enzymes It remains unclear to what extent metabolism contributes to epigenetic regulation in stem cells or the degree to which metabolic changes during differentiation contribute to alterations in chromatin structure and gene expression. However, the activities of chromatin-modifying enzymes are known to be metabolically regulated because metabolites serve as cofactors for them [79] (Fig. 3). Acetyl-CoA levels regulate histone acetylation in yeast and mammalian cells [80, 81]. In mammalian cells, acetyl-CoA for histone acetylation is generated from citrate by ATP citrate lyase [82]. Increased histone acetylation after growth factor stimulation or differentiation in cultured adipocytes depends on ATP citrate lyase and glucose [82]. Knockdown of ATP citrate lyase induces the differentiation of cultured myoblasts [83]. Sirtuins regulate stem cell self-renewal and differentiation in multiple tissues by diverse mechanisms, including the regulation of gene expression [84–86], mitochondrial metabolism [87], nuclear translocation of p53 [88] and genomic integrity [89]. Sirtuins deacetylate lysines on many proteins in a manner that depends on NAD+ [90]. During murine skeletal muscle differentiation, the [NAD+]/ [NADH] ratio decreases [91]. Increasing this ratio by treating myoblasts with pyruvate blocks differentiation, whilst decreasing the ratio by treating the cells with lactate promotes differentiation. Overexpression of Sirt1 blocks differentiation in a manner dependent on its deacetylase activity [91]. Glucose restriction in cultured skeletal muscle progenitors activates AMPK, leading to increased nicotinamide phosphoribosyltransferase activity, an increase in the [NAD+]/[NADH] ratio and inhibition of myogenesis [92]. Myogenesis is rescued by Sirt1 knockdown, suggesting that AMPK/Sirt1 inhibits myoblast differentiation when nutrients

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Key Symposium: Stem cell metabolism

Chromatin Modification GlcNAc

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Fig. 3 Metabolic intermediates regulate chromatin-modifying enzyme activity and gene expression. Histone proteins are subject to modification, including acetylation, methylation and O-GlcNAcylation [90, 95, 108], which in turn contributes to changes in chromatin structure and gene expression. The chromatin-modifying enzymes that catalyse these reactions require metabolic intermediates as cofactors. Histone acetylation is catalysed by lysine acetyltransferases in a reaction dependent on acetyl-CoA [82]. Sirtuin histone deacetylases require nicotinamide adenine dinucleotide (NAD+) to remove acetyl marks from lysine residues [90]. Histone methylation is catalysed by lysine methyltransferases, requiring S-adenosylmethionine (SAM). Histone methylation is removed by histone demethylases, including lysine demethylase 1 (Lsd1), which requires flavin adenine dinucleotide (FAD), and members of the Jumonji C family, which require the tricarboxylic acid (TCA) cycle metabolite a-ketoglutarate, Fe(II) and O2 [96]. O-GlcNAcylation of histones is catalysed by O-GlcNAc transferase and requires urine diphosphate-N-acetylglucosamine (UDP-GlcNAc) [108]. The metabolic intermediate, a-ketoglutarate, also regulates the ten-eleven translocation (TET) family of DNA hydroxylases, which catalyses the conversion of 5-methylcytosine to 5-hydroxymethylcytosine on DNA. These chromatin-modifying enzymes are regulated, in part, by the available levels of their cofactors, although it remains unclear to what extent physiological variations in the cofactors impact epigenetic regulation in stem cells. The metabolic intermediates that function as cofactors are indicated in red.

are scarce [92]. Lysine acetylation and deacetylation also occur on nonhistone proteins such as regulators of chromatin remodelling, cell cycle, RNA splicing, nuclear transport and metabolism [93, 94]. To understand the function of acetylation in stem cells, it will be critical to investigate how acetylation differs in stem cells compared with progenitors, the extent to which these changes take place on histones versus other proteins and the mechanisms by which metabolism regulates protein acetylation in stem cells. The regulation of chromatin-modifying enzymes by metabolite cofactors is not limited to protein lysine acetyltransferases. Histone and DNA methyltransferase enzymes transfer methyl groups from SAM to lysine residues of proteins or cytosine residues of DNA, altering chromatin structure and gene expression [95]. SAM is derived from methionine

and regenerated by methyl groups generated through threonine, glycine, serine and folate metabolism (Fig. 2). As already discussed, defects in threonine metabolism in mouse ES cells decrease global histone H3 lysine 4 trimethylation as a result of decreased SAM levels [24]. Dioxygenase enzymes, including ten-eleven translocation (TET) DNA hydroxylases and Jumonji C family histone demethylases, use a-ketoglutarate as a cofactor for the reactions they catalyse [96]. The metabolite a-ketoglutarate is generated from isocitrate in the TCA cycle as well as from glutamate. Therefore, metabolic regulation of a-ketoglutarate levels may regulate stem cell differentiation through changes in histone and/or DNA methylation. Consistent with this possibility, point mutations in isocitrate dehydrogenase (IDH, which converts isocitrate to a-ketoglutarate) in gliomas, ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 12–24

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acute myeloid leukaemias and other cancers lead to production of 2-hydroxyglutarate, which inhibits dioxygenase enzymes [97, 98]. Mice expressing the IDH1 R132H mutant allele in haematopoietic cells have increased numbers of haematopoietic progenitors and impaired differentiation, potentially as a result of aberrant DNA and histone methylation [99, 100] or modulation of prolyl hydroxylases [98, 100]. Expression of the IDH2 R172K mutant allele inhibits adipocyte differentiation in vitro and represses adipocyte-specific gene expression, possibly as a result of increased histone methylation [101]. Studies in cancer cells have shown that alterations in TCA cycle metabolites such as fumarate and succinate also regulate DNA and histone demethylases of the dioxygenase family through competitive inhibition of a-ketoglutarate binding [102]. Histone lysine-specific demethylase 1 (LSD1) represses gene expression by demethylating monoand dimethylated histone H3 lysines 4 and 9 [103]. Lsd1 uses flavin adenine dinucleotide (FAD) as a cofactor [103], and disruption of FAD production leads to increased expression of some LSD1 target genes [104]. Lsd1 regulates the differentiation of ES cells and HSCs [105–107]; however, the extent to which Lsd1 is metabolically regulated in these stem cells is unclear. Epigenetic regulation in stem cells is also influenced by the production of uridine diphosphate-Nacetylglucosamine (UDP-GlcNAc) in the hexosamine biosynthetic pathway, a branch of glucose metabolism [108]. The enzyme O-GlcNAc transferase (OGT) employs UDP-GlcNAc in the O-GlcNAcylation of serine and threonine residues of numerous substrates [108], including histones [109]. The pluripotency factors Oct4 and Sox2 are O-GlcNAcylated, which regulates their function in pluripotent stem cells [110]. Complete loss of OGT results in ES cell death, whilst reduced OGT expression disrupts ES cell self-renewal and inhibits the reprogramming of fibroblasts to iPS cells [110, 111]. Reprogramming of mouse embryonic fibroblasts in low-glucose medium, which reduces O-GlcNAc levels, results in fewer iPS cell colonies, raising the possibility that OGT activity is metabolically regulated in stem cells [110]. Nonetheless, it remains uncertain whether variations in UDPGlcNAc levels under physiological conditions in vivo are sufficient to alter the activities of epigenetic regulators or to change gene expression in stem cells. 20

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Key Symposium: Stem cell metabolism

Nutrition regulates stem cell function Stem cell function is modulated in response to nutritional changes. Protein deprivation in Drosophila inhibits ovarian stem cell proliferation as a consequence of decreases in insulin-like peptide signalling [112]. Reduced insulin signalling influences stem cells directly [113] and reduces the number of cap cells in the niche [114]. In the Drosophila intestine and testis, protein starvation reduces stem cell number and proliferation, also by modulating insulin/insulin-like growth factor signalling, without compromising the ability of stem cells to repopulate the tissues when nutrition is restored [17]. Nutrient levels are not just permissive for stem cell function but also determine at least certain aspects of stem cell function. Drosophila neuroblasts become quiescent at the end of embryonic development followed by reactivation in response to dietary amino acids when larvae start eating [115, 116]. Fat body cells sense amino acids and stimulate secretion of insulin-like peptides from glia in the neuroblast niche, promoting neuroblast proliferation [115, 116]. Although these studies suggest that stem cells undergo reversible changes in response to changes in nutrition, irreversible changes do occur, such as blood progenitors of the fly lymph gland differentiating when larvae are starved [117, 118]. The effects of caloric restriction on vertebrate stem cells are also beginning to be investigated. Caloric restriction increases longevity and reduces morbidities associated with ageing [119]. In line with this idea, caloric restriction delays the decline in HSC function that is observed with age, although the effect may vary amongst mouse strains [120]. Caloric restriction enhances the function and frequency of skeletal muscle stem cells [19]. In the intestinal system, caloric restriction increases the number of intestinal epithelial stem cells by stimulating the number and function of the Paneth cells that form the intestinal stem cell niche [16]. In contrast, fasting in Drosophila reduces intestinal stem cell number and proliferation and decreases the size of the gut [121]. The onset of feeding increases the number of stem cells, their activity and total gut size [121]. All these results indicate that stem cells and their niche sense nutritional status and modulate their function in response. How stem cells adjust intracellular metabolism in response to these physiological challenges is a major open question.

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Conclusions and perspective Intracellular metabolism provides energy and biosynthetic molecules for the cell to carry out its functions. Given the functional differences between stem cells, restricted progenitors and differentiated cells, it is not surprising that these cells exhibit differences in metabolism and metabolic regulation. Nonetheless, a great deal of additional study will be required to address the degree to which metabolism differs in stem cells versus restricted progenitors and the extent to which metabolic changes influence cell fate determination and other aspects of stem cell identity and function. A more comprehensive picture of stem cell metabolism could allow us to rationally develop culture conditions that promote stem cell maintenance or expansion for cell therapy. Moreover, monitoring endogenous stem cell metabolism in vivo using tools such as fluorescent probes or metabolomics on uncultured cells will help us to understand metabolic regulation under physiological conditions and how it must change in response to injury. Metabolism must be coordinately regulated with other stem cell self-renewal mechanisms, but little is known about how these pathways integrate. For example, metabolic control integrates with cell cycle control in yeast [81], but minimal data on this exist for mammalian stem cells. Similarly, the activation of signal transduction pathways such as the PI3-kinase pathway is recognized as having widespread effects on both stem cell function [122] and metabolism [123]. However, it is not clear to what extent the metabolic changes mediate the effects of these signals on stem cells. In addition, stem cells reside in specialized niches, but it has not been determined whether signals from the niche support stem cell metabolism. As these mechanisms are clarified, we will gain a much more complete understanding of tissue homoeostasis as well as the extent to which metabolic pathways are regulated differently in various cell types. Conflict of interest statement No conflicts of interest were declared. Acknowledgements SJM is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics and the director of the Hamon Laboratory for Stem Cells and Cancer. This work

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was supported by the Cancer Prevention and Research Institute of Texas, the National Institute on Aging (R37 AG024945) and the National Institute of Neurological Disorder and Stroke (NS040750). MA is a Royal Commission for the Exhibition of 1851 Research Fellow. RJB is supported by a Ruth L. Kirschstein National Research Service Award. We apologize to those authors whose work was not cited due to space constraints.

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Metabolic regulation of stem cell function.

Stem cell function is regulated by intrinsic mechanisms, such as transcriptional and epigenetic regulators, as well as extrinsic mechanisms, such as s...
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