Pluripotent stem cell energy metabolism: an update
2014; Springer Nature; Volume: 34; Issue: 2 Linguagem: Inglês
10.15252/embj.201490446
ISSN1460-2075
AutoresTara TeSlaa, Michael A. Teitell,
Tópico(s)Genomics and Rare Diseases
ResumoReview4 December 2014free access Pluripotent stem cell energy metabolism: an update Tara Teslaa Tara Teslaa Molecular Biology Institute, University of California, Los Angeles, CA, USA Search for more papers by this author Michael A Teitell Corresponding Author Michael A Teitell Molecular Biology Institute, University of California, Los Angeles, CA, USA Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA Department of Bioengineering, University of California, Los Angeles, CA, USA Department of Pediatrics, University of California, Los Angeles, CA, USA California NanoSystems Institute, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA Search for more papers by this author Tara Teslaa Tara Teslaa Molecular Biology Institute, University of California, Los Angeles, CA, USA Search for more papers by this author Michael A Teitell Corresponding Author Michael A Teitell Molecular Biology Institute, University of California, Los Angeles, CA, USA Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA Department of Bioengineering, University of California, Los Angeles, CA, USA Department of Pediatrics, University of California, Los Angeles, CA, USA California NanoSystems Institute, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA Search for more papers by this author Author Information Tara Teslaa1 and Michael A Teitell 1,2,3,4,5,6,7 1Molecular Biology Institute, University of California, Los Angeles, CA, USA 2Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA 3Department of Bioengineering, University of California, Los Angeles, CA, USA 4Department of Pediatrics, University of California, Los Angeles, CA, USA 5California NanoSystems Institute, University of California, Los Angeles, CA, USA 6Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA 7Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA *Corresponding author. Tel: +1 310 206 6754; Fax: +1 310 267 0382; E-mail: [email protected] The EMBO Journal (2015)34:138-153https://doi.org/10.15252/embj.201490446 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage-directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications. Glossary 5hmc 5-hydroxymethylcytosine 5mC 5-methylcytosine ADP adenosine diphosphate AMD1 adenosylmethionine decarboxylase 1 AMP adenosine monophosphate AMPK AMP-activated protein kinase ARNT aryl hydrocarbon receptor nuclear translocator ATP adenosine triphosphate Cited2 CREB-binding protein (CBP)/p300-interacting transactivator with glutamic acid and aspartic acid tail 2 DEPTOR DEP domain-containing mTOR-interacting protein Drp1 dynamin-related protein 1 EBs embryoid bodies ETC electron transport chain FAD flavin adenosyl dinucleotide FAO fatty acid oxidation FBS fetal bovine serum HAT histone acetyltransferase hESCs human embryonic stem cells HIF1α hypoxia-inducible factor 1α HIF1β hypoxia-inducible factor 1β HIF2α hypoxia-inducible factor 2α hiPSCs human-induced pluripotent stem cells hLIF human leukemia inhibitory factor HMT histone methyltransferase hPSCs human pluripotent stem cells IMS intermembrane space iPSCs induced pluripotent stem cells JmjC Jumonji domain-containing Jph2 junctophilin 2 KLF4 Kruppel-like factor 4 LKB1 liver kinase B1 LSD1 lysine-specific demethylase 1 MEFs mouse embryonic fibroblasts mEpiSCs mouse epiblast stem cells mESCs mouse embryonic stem cells MET mesenchymal-to-epithelial transition Mfn1 mitofusin-1 Mfn2 mitofusin-2 miPSCs mouse-induced pluripotent stem cells MOMP mitochondrial outer membrane permeabilization mPSCs mouse pluripotent stem cells mPTP mitochondrial permeability transition pore mtDNA mitochondrial DNA mTOR mammalian target of rapamycin mTORC1 mammalian target of rapamycin complex 1 mTORC2 mammalian target of rapamycin complex 2 NAD nicotinamide adenine dinucleotide NPCs neural progenitor cells NuRD nucleosome remodeling and deacetylase OCT4 octamer-binding protein 4 OPA1 optic atrophy 1 OXPHOS oxidative phosphorylation PC pyruvate carboxylase PDH pyruvate dehydrogenase PDK1 pyruvate dehydrogenase kinase 1 PDK3 pyruvate dehydrogenase kinase 3 Phb2 prohibitin 2 PHD prolyl hydroxylase POU5F1 POU class 5 homeobox 1 PSCs pluripotent stem cells pVHL von Hippel-Lindau tumor suppressor protein PYGL glycogen phosphorylase liver REX1 reduced expression 1 ROS reactive oxygen species SAM s-adenosyl methionine SOX2 SRY (sex-determining region Y)-box 2 TCA cycle tricarboxylic acid cycle TDH threonine dehydrogenase Tet1 ten–eleven translocation Tsc2 tuberous sclerosis 2 UCP2 uncoupling protein 2 Introduction Energy production in early mammalian development depends upon many factors, including substrate availability, uptake, and O2 tension. All mammalian cells produce ATP by differing proportions of glycolysis and oxidative phosphorylation (OXPHOS), with the balance between these processes at specific developmental stages or states of cellular activation controlled by multiple intra- and extracellular factors. Glycolysis is the enzymatic conversion of glucose to pyruvate, which generates 2 net ATP molecules per molecule of glucose. Cells that depend mainly on glycolysis for ATP production further convert pyruvate to lactate, which is excreted. By contrast, cells in oxygen-rich environments may prefer OXPHOS for more efficient ATP production, which on average nets 34 additional ATP molecules per glucose by oxidizing pyruvate to acetyl-CoA in the mitochondrial tricarboxylic acid (TCA) cycle. During pre-implantation development of early mouse embryos, ATP is produced mainly by OXPHOS from uptake of pyruvate, lactate, amino acids, and triglyceride-derived fatty acids (Brinster & Troike, 1979; Martin & Leese, 1995; Jansen et al, 2008; Leese, 2012). This is followed by a shift to a more balanced mixture of glycolysis and OXPHOS with increasing glucose uptake in the low O2 microenvironment of an implanting blastocyst (Leese & Barton, 1984; Houghton et al, 1996; Zhou et al, 2012). In vitro studies report a similar increase in glucose uptake in early human embryos advancing to the blastocyst stage in a dish (Gardner et al, 2001). Pyruvate and glucose uptake and amino acid turnover are predictors of human blastocyst quality and enhanced viability for in vitro fertilization protocols (Houghton et al, 2002; Brison et al, 2004). In concept, in vivo differences in early mammalian embryo energy metabolism should be replicated in vitro by cells obtained from distinct stages of embryonic development that are maintained in similar culture conditions. Human embryonic stem cells (hESCs) originate from the blastocyst inner cell mass and hold great clinical potential for cell replacement therapies because of their high proliferative capacity and their ability to differentiate into any cell type in the body (Thomson et al, 1998). However, the clinical use of differentiated hESCs is limited by ethical concerns regarding the method of hESC acquisition and by potential allogeneic immune rejection (Zhao et al, 2011). To help circumvent these issues, mammalian somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) through ectopic expression of different combinations of transcription factors, such as the “Yamanaka cocktail” of POU5F1, SOX2, KLF4, and MYC (Takahashi & Yamanaka, 2006; Takahashi et al, 2007) or by other methods (Yu et al, 2007; Huangfu et al, 2008; Lowry et al, 2008; Ichida et al, 2009; Kim et al, 2009; Lin et al, 2009; Lyssiotis et al, 2009; Zhou et al, 2009a; Jia et al, 2010; Warren et al, 2010; Zhu et al, 2010; Anokye-Danso et al, 2011; Hu et al, 2011; Miyoshi et al, 2011; Bayart & Cohen-Haguenauer, 2013; Hou et al, 2013; Sommer & Mostoslavsky, 2013). Both hESCs and human iPSCs (hiPSCs) are markedly glycolytic, secreting abundant lactate, in ambient (~160 mm Hg) O2 (Zhang et al, 2011; Zhou et al, 2012), which differs substantially from the ~40 mm Hg O2 partial pressure measured for several mammalian reproductive tracts (Fischer & Bavister, 1993). A similar glycolytic preference in different O2 environs at first pass suggests a pluripotent stage-specific metabolic program that is relatively insensitive to O2 levels in chemically defined or undefined culture media. However, hESCs replicate well in 1–5% O2 and resist spontaneous differentiation compared to culture in 21% O2, suggesting that O2 levels influence the factors that maintain pluripotency (Ezashi et al, 2005). Somatic cell reprogramming to hiPSCs or mouse iPSCs (miPSCs) requires a shift from mainly OXPHOS to mainly glycolytic metabolism and high levels of lactate production (Yoshida et al, 2009; Zhou et al, 2012). iPSC production efficiency is enhanced by performing reprogramming in hypoxia or inducing a shift to glycolysis during this process, indicating a role for metabolism in controlling and not just passively responding to de-differentiation (Yoshida et al, 2009; Zhu et al, 2010; Jung et al, 2013). In fact, a shift to glycolysis may occur early in reprogramming before self-renewal and pluripotent gene expression (Folmes Clifford et al, 2011; Mathieu et al, 2014; Prigione et al, 2014). Interestingly, glycolysis-skewed pluripotent stem cells (PSCs), which include ESCs and iPSCs, resemble many cancer cell types that revert to “Warburg metabolism” (aka “aerobic glycolysis”) upon malignant transformation and coupled cellular de-differentiation (Warburg, 1956; Christofk et al, 2008; Figueroa et al, 2010; Lu et al, 2012; Ward Patrick & Thompson Craig, 2012). OXPHOS is low in hPSCs, which includes both hESCs and hiPSCs, and the mitochondria are perinuclear and less fused into a filamentous network structure with swollen, less mature appearing inner membrane cristae folds than mitochondria in terminally differentiated cell types (Oh et al, 2005; St John et al, 2005, 2006; Houghton, 2006; Suhr et al, 2010; Zeuschner et al, 2010; Folmes Clifford et al, 2011; Zhang et al, 2011). The perinuclear arrangement of mitochondria has also been noted in cleavage stage embryos of several mammalian species including mice and humans and has been suggested as a “stemness” property (Batten et al, 1987; Barnett et al, 1996; Wilding et al, 2001; Squirrell et al, 2003; Lonergan et al, 2006, 2007). Mouse ESCs (mESCs), which like hESCs are obtained from the blastocyst inner cell mass, contain mitochondria that display even less mature morphological and ultrastructural features than hPSCs (Folmes Clifford et al, 2011; Zhou et al, 2012). However, hPSCs metabolically resemble developmentally more mature, glycolytic mouse epiblast stem cells (mEpiSCs), obtained from the post-implantation epiblast, instead of mESCs, which show a bivalent metabolism that can switch between glycolysis and OXPHOS on demand (Zhou et al, 2012). This metabolic comparison is consistent with biomarker and functional features of standard laboratory hPSCs that are “primed”, or more mature, than naïve, or ground state hPSCs. Naïve hPSCs, similar to mESCs that represent the least mature pluripotent stage, have recently been obtained by hPSC exposure to chemical inhibitor and growth factor cocktails or by transient expression of two transcription factors combined with two chemical inhibitors and human leukemia inhibitory factor (Fig 1) (Gafni et al, 2013; Takashima et al, 2014; Theunissen Thorold et al, 2014; Ware et al, 2014). hPSCs reset to a naïve state through transient ectopic expression of NANOG and KLF4 respire at a higher level than “primed” hPSCs, similar to pre-implantation mouse embryos and naïve mESCs (Fig 1) (Takashima et al, 2014). The regulation of energy metabolism therefore appears intertwined with genetic and epigenetic mechanisms that control PSC maturation state through pathways that require further elucidation. Figure 1. Influence of energy metabolism on pluripotent statusNaïve human pluripotent stem cells (hPSCs) show an increase in ATP production through oxidative phosphorylation (OXPHOS) compared to more mature, “primed” hPSCs. Primed hPSCs can be converted to the naïve state through ectopic expression of NANOG and KLF4, inhibition of the ERK pathway by two inhibitors (2i), and stimulation with human leukemia inhibitory factor (L) (Takashima et al, 2014). Alternatively, the naïve state can be induced with a cocktail of five inhibitors and growth factors Activin and hLIF (5i/L/A) (Theunissen Thorold et al, 2014). Somatic cells can be reprogrammed with OCT4, SOX2, KLF4, and c-MYC (OSKM). Fibroblasts are more oxidative than primed hPSCs. Factors that activate glycolysis and inhibit OXPHOS promote induced PSC (iPSC) reprogramming. Vitamin C enhances iPSC reprogramming as an antioxidant and as a cofactor for epigenetic enzymes. Rapamycin, an inhibitor of the mTOR pathway, also increases the efficiency of iPSC reprogramming. Withdrawal of methionine from hPSC culture, which is required to maintain DNA and histone methylation, promotes differentiation. Download figure Download PowerPoint Metabolic regulation of self-renewal, reprogramming, and differentiation Reprogrammed iPSCs maintain an “epigenetic memory” or chromatin signature of the cells from which they were generated that can impact their re-differentiation potential and function (Kim et al, 2010, 2011; Bar-Nur et al, 2011). Changes in cellular metabolism can impact the activity of epigenome-modifying enzymes, as discussed below (Kaelin William & McKnight Steven, 2013). Therefore, manipulation of culture conditions could erase or generate new epigenetic marks during iPSC reprogramming, PSC differentiation, or steady-state growth that will affect the functional potential of the end resulting cell. For therapeutic utility, identifying specific, reproducible, and chemically defined culture conditions to produce safe and functional differentiated cells from hPSCs, or by transdifferentiation protocols, will be required. Several key cell types targeted for cell replacement therapies have high energy demands, such as cardiomyocytes and neurons. Therapeutic applications will therefore require re-establishment of a cell type-specific, fully functional mitochondrial network to support the energy and other mitochondrial supplied factors for these replacement cell types. Importantly, mitochondrial dysfunction due to impaired nucleus and mitochondrial encoded genes has been linked to > 400 named human diseases, including multiple neurodegenerative disorders and cancer (Nunnari & Suomalainen, 2012). The discovery that hypoxia maintains self-renewal and increases the efficiency of reprogramming to pluripotency has stimulated studies to determine the role of oxygen tension in cell fate determination. A striking difference in mitochondrial morphology between PSCs and their differentiated derivatives has similarly spurred studies to decipher the mechanisms that control cell state-specific mitochondrial structure and function. Adding to this complexity are state-specific levels of cellular metabolites, such as the AMP/ATP ratio and amino acid availabilities, which can impact PSC gene expression and cell function. In the first part of this review, the effect that these components of cellular metabolism have on self-renewal and differentiation is explored. Oxygen tension and hypoxia-inducible factors (HIFs) Reduced O2 (1–5%) can be used for hPSC tissue culture to mimic the hypoxic early embryonic microenvironment in vivo. Transcription factors such as hypoxia-inducible factor 1α (HIF1α) and 2α (HIF2α) control the genomic response to low O2 tension by promoting the expression of genes such as pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA), and glycogen phosphorylase liver (PYGL), which encode for key glycolysis regulating enzymes (Greer et al, 2012; Zhou et al, 2012). HIF1α and HIF2α are degraded in ambient air (~21% O2 at sea level) by hydroxylation and ubiquitination from O2-dependent prolyl hydroxylases (PHDs) and the von Hippel-Lindau tumor suppressor protein (pVHL), respectively (Maxwell et al, 1999; Ohh et al, 2000; Jaakkola et al, 2001). In hypoxia, HIF1α and HIF2α are stabilized and form a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT; aka HIF1β). HIF heterodimers accumulate in the nucleus and bind to promoters of genes that regulate cellular adaptation to hypoxia, stimulating their transcription (Wang et al, 1995). As noted above, hypoxia inhibits the spontaneous differentiation of hESCs and also increases the efficiency of iPSC reprogramming (Ezashi et al, 2005; Yoshida et al, 2009). HIF2α-dependent transactivation of Oct4 gene expression promotes self-renewal and the maintenance of pluripotency in hypoxia (Niwa et al, 2000; Covello et al, 2006). HIF1α stabilization promotes a metabolic shift to increased glycolysis and lactate production during the transition from mESCs to mEpiSCs. Ectopic expression of a non-degradable form of HIF1α in mESCs is sufficient to induce a mEpiSC-like phenotype with a decrease in OXPHOS and an increase in glycolysis, indicating the importance of HIF transcription factors in early embryonic development (Zhou et al, 2012). Hypoxia causes the re-entry of lineage-committed progenitor cells derived from hESCs back into pluripotency. hESCs transiently induced to lineage non-specific differentiation by fetal bovine serum (FBS) addition instead de-differentiate in 2% O2, whereas hESCs differentiated with FBS in air (~21% O2) proceed ahead. As expected, differentiated hESCs shift their metabolic balance from mainly glycolysis to OXPHOS, whereas de-differentiated hESCs remain glycolytic. De-differentiated hESCs are enriched for HIF1α and HIF2α target gene expression, suggesting a role for HIFs in promoting re-entry into the pluripotent state (Mathieu et al, 2013). HIF2α is required early in iPSC reprogramming for shifting from mainly OXPHOS to lactate-producing glycolysis, but stabilization of HIF2α beyond day 12 of reprogramming is detrimental (Fig 1). Ectopic expression of sequence-stabilized HIF1α and/or HIF2α is sufficient to impair OXPHOS in fibroblasts (Mathieu et al, 2014). HIF1α improves iPSC reprogramming efficiency by increasing glycolysis and lactate production through activation of target genes PDK1, pyruvate dehydrogenase kinase 3 (PDK3), and pyruvate kinase isoform M2 (PKM2) (Fig 1) (Mathieu et al, 2014; Prigione et al, 2014). CREB-binding protein (CBP)/p300-interacting transactivator with glutamic acid and aspartic acid tail 2 (Cited2) is a HIF1α antagonist. Cited2 is expressed and inhibits HIF1α during lineage non-specific mESC differentiation, with Cited2 knockout mESCs unable to silence Oct4 or activate differentiation-related genes. shRNA knockdown of HIF1α in Cited2-deficient mESCs partially rescues this defect in lineage non-specific differentiation (Li et al, 2014). HIF transactivation also regulates the lineage-specific differentiation of human neural progenitor cells (NPCs) (Xie et al, 2014). Neurons are more oxidative than glial cells (Kasischke et al, 2004; Bélanger et al, 2011). NPCs generated by changing the hPSC culture media to enable the formation of rosette structures can develop into neurons or glia by directed NPC differentiation. NPCs derived from hESC differentiation are mainly glycolytic (Birket et al, 2011), and proteomic comparisons of hESCs at different stages of neural lineage differentiation show differential expression of enzymes that regulate redox homeostasis (Fathi et al, 2014). The differentiation of mixed lineage embryoid bodies (EBs) in 2% O2 also promotes neurogenesis. Most cells differentiated from hPSC-derived NPCs are neurons with a concomitant small number of glial cells. Remarkably, just shifting the O2 environment during NPC differentiation from 21 to 2% O2 strongly shifts the culture toward gliogenesis and away from neurogenesis. This effect can be replicated with HIF stabilizing deferoxamine in ambient O2 as well. HIF1α promotes gliogenesis through the inhibition of LIN28a by displacement of MYC on the LIN28 promoter. Remarkably, even a transient low O2 period during NPC differentiation skews the resulting culture strongly toward gliogenesis, suggesting that the O2-sensing machinery induces a lasting effect on NPC differentiation potential (Xie et al, 2014). Mitochondria and the electron transport chain Mammalian cells consume glucose and convert it to pyruvate with ATP production in several enzymatic steps during glycolysis (TeSlaa & Teitell, 2014). Pyruvate in turn can be converted to lactate by LDH and will be excreted from the cell. Alternatively, pyruvate can enter mitochondria as acetyl-CoA, through the action of pyruvate dehydrogenase (PDH), or as oxaloacetate, via pyruvate carboxylase (PC), to generate CO2 and additional ATP through OXPHOS. Pyruvate that enters the TCA cycle regenerates NADH and FADH2, which subsequently donate electrons to the electron transport chain (ETC) and establish a hydrogen ion gradient, which is used by the F0F1 ATP synthase to make ATP from ADP plus inorganic phosphate. ETC activity therefore depends on the ADP/ATP ratio as well as the levels of environmental and internal resources that include TCA cycle carbon substrates and electron acceptors. The levels and functional assemblies of nucleus and mitochondrial DNA (mtDNA) encoded ETC subunits that comprise ETC complexes I through V, excluding nucleus-encoded complex II, and their assemblies into higher order supercomplexes, along with mitochondrial network fusion/fission status, further determines the minimal and maximal respiratory potential of most cells, which remains to be established for PSCs. The complexity of ETC complex regulation has been shown in other systems, including the differential expression of subunits in complex IV and the assembly of complexes I, III, and IV into supercomplexes (Fukuda et al, 2007; Chen et al, 2012b; Ikeda et al, 2013; Lapuente-Brun et al, 2013). Low-level respiration and activity of the ETC in primed PSCs, including hPSCs and mEpiSCs, may at least partially result from the hypoxic microenvironment in vivo, immediately post-implantation. In addition, the donation of electrons from NADH and FADH2 to the ETC may result in oxidative stress through formation of reactive oxygen species (ROS). Primed PSCs may limit ROS to prevent damage to proteins, lipids, and importantly DNA within the cell. However, steady-state ROS levels also increase with PSC differentiation and can help drive differentiation at later stages of precursor cell development (Cho et al, 2006; Saretzki et al, 2008). The addition of antioxidants to the culture medium of hiPSCs enhances their genomic stability, consistent with a benefit for maintaining low ROS in PSCs (Luo et al, 2014). Vitamin C, an antioxidant, also enhances the efficiency of iPSC reprogramming (Esteban et al, 2010), although vitamin C may also impact reprogramming efficiency through epigenetic mechanisms described below. ATP and ROS production by OXPHOS is further limited by several mechanisms in hPSCs, such as by the expression of uncoupling protein 2 (UCP2) (Zhang et al, 2011). UCP2 transports four carbon TCA cycle intermediates out of the mitochondria, effectively reducing carbon substrates for use in OXPHOS (Vozza et al, 2014). Also, nuclear genes encoding multiple subunits of cytochrome C oxidase (complex IV of the ETC), which donates electrons to O2, are expressed at a lower level in mEpiSCs compared to mESCs (Zhou et al, 2012). DMSO-induced differentiation of mPSCs increases ETC complex I and complex IV activities along with mitochondrial biogenesis to support an increase in mitochondrial ATP production (Han et al, 2014). The ETC maintains the mitochondrial inner membrane electrochemical potential, Δψ, which is required to prevent mitochondrial outer membrane permeabilization (MOMP) and the release of proapoptotic intermembrane space (IMS) proteins, such as cytochrome c, that induce apoptosis (Green & Kroemer, 2004). When ETC activity is low, Δψ can be additionally supported by the hydrolysis of ATP in the complex V ATP synthase, which results in the translocation of protons from the mitochondrial matrix to the IMS to increase Δψ (Hatefi, 1985). hPSCs have relatively low respiration and ETC activity; therefore, ATP hydrolase activity of the ATP synthase helps to maintain Δψ and sustain cell viability (Zhang et al, 2011). Interestingly, hPSCs maintain a higher Δψ than their differentiated derivatives (Chung et al, 2007; Armstrong et al, 2010; Prigione et al, 2011), which has been proposed to enable rapid metabolic changes during differentiation (Folmes Clifford et al, 2012b; Folmes et al, 2012a) and possibly to maintain a fragmented mitochondrial network (Mattenberger et al, 2003). iPSC reprogramming of mouse embryonic fibroblasts (MEFs) causes major changes in the expressed proteome in two stages. ETC complex I and complex IV proteins are reduced early during reprogramming, in contrast to components of ETC complexes II, III, and V, which are transiently induced during a second, intermediate reprogramming phase (Hansson et al, 2012). The efficiency and speed of iPSC reprogramming is enhanced when OXPHOS is decreased by inhibition of any of the ETC respiratory complexes, consistent with a required shift toward glycolysis (Fig 1) (Son et al, 2013b). An increase in OXPHOS capacity is required for proper cardiomyocyte lineage-directed differentiation from PSCs. Cardiomyocyte differentiation induces the expression of nucleus-encoded genes for mtDNA transcription factors, mtDNA replication factors, components of the fatty acid oxidation (FAO) machinery, enzymes of the TCA cycle, and ETC subunits (St John et al, 2005; Chung et al, 2007; Tohyama et al, 2013). Cardiomyocyte-directed differentiation is enhanced by the generation of ROS by NADPH oxidase-like enzymes (Sauer et al, 2000; Crespo et al, 2010). Agonists of peroxisome proliferator-activated receptor α (PPARα), a highly expressed nuclear hormone receptor in the heart associated with FAO, promote cardiomyogenesis of mESCs through increasing ROS production (Sharifpanah et al, 2008). Differences in carbon substrate types can be used to purify metabolically mature mouse cardiomyocytes following differentiation from mPSCs because of key differences in metabolite handling capacity between mouse cardiomyocytes and mPSCs. Fetal cardiomyocytes preferentially consume lactate for the production of ATP (Fisher et al, 1981; Werner & Sicard, 1987). Therefore, cardiomyocytes derived in vitro from PSCs can utilize lactate in the absence of glucose to produce ATP, whereas mESCs and MEFs are unable to use lactate for ATP production. When cultured in glucose-free media supplemented with lactate, functional mouse cardiomyocytes can be recovered at ~99% purity (Tohyama et al, 2013). Mitochondrial dynamics The dynamic fusion and fission/fragmentation of an interlacing mitochondrial network enables mixing of mitochondrial contents and the degradation of damaged mitochondria to maintain robust energy and metabolite production (Twig et al, 2008; Westermann, 2012). Mitochondrial network fusion status is a determinant of maximal respiratory capacity (Chen et al, 2005; Yu et al, 2006). PSCs show a punctate, fragmented mitochondrial network that progressively fuses during differentiation, which increases respiratory capacity (Zhang et al, 2011). The GTPase dynamin-related protein 1 (DRP1), which causes mitochondrial fission, can be inhibited to induce a fused mitochondrial network. Pharmacological inhibition of Drp1 to maintain a fused mitochondrial network inhibits iPSC reprogramming (Vazquez-Martin et al, 2012a), although shRNA knockdown of Drp1, also resulting in mitochondrial fusion, did not impair iPSC reprogrammi
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