Mitochondrial dynamics and metabolism in induced pluripotency

Journal Pre-proof Mitochondrial dynamics and metabolism in induced pluripotency

Javier Prieto, Xavier Ponsoda, Juan Carlos Izpisua Belmonte, Josema Torres PII:

S0531-5565(19)30677-1

DOI:

https://doi.org/10.1016/j.exger.2020.110870

Reference:

EXG 110870

To appear in:

Experimental Gerontology

Received date:

1 October 2019

Revised date:

20 December 2019

Accepted date:

5 February 2020

Please cite this article as: J. Prieto, X. Ponsoda, J.C.I. Belmonte, et al., Mitochondrial dynamics and metabolism in induced pluripotency, Experimental Gerontology(2020), https://doi.org/10.1016/j.exger.2020.110870

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© 2020 Published by Elsevier.

Journal Pre-proof

Mitochondrial dynamics and metabolism in induced pluripotency

Javier Prieto1,2* , Xavier Ponsoda1,3, Juan Carlos Izpisua Belmonte2 and Josema Torres1,3,*

Departamento Biología Celular, Biología Funcional y Antropología Física, Universitat de

València, Calle Dr. Moliner 50, 46100 Burjassot, Spain

Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey

Pines Road, La Jolla, CA 92037, USA

Instituto de Investigación Sanitaria (INCLIVA), Avenida de Menéndez y Pelayo 4, 46010

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Valencia, Spain

* Correspondence should be addressed to Javier Prieto ([email protected]) and Josema

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Summary

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Torres ([email protected])

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Somatic cells can be reprogrammed to pluripotency by either ectopic expression of defined factors or exposure to chemical cocktails. During reprogramming, somatic cells undergo dramatic changes in a wide range of cellular processes, such as metabolism, mitochondrial morphology and function, cell signaling pathways or immortalization. Regulation of these processes during cell reprograming lead to the acquisition of a pluripotent state, which enables indefinite propagation by symmetrical self-renewal without losing the ability of reprogrammed cells to differentiate into all cell types of the adult. In this review, recent data from different laboratories showing how these processes are controlled during the phenotypic transformation of a somatic cell into a pluripotent stem cell will be discussed.

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Journal Pre-proof Introduction Stem cells are a particular type of cells capable to self-renew and differentiate. Embryonic stem cells (ESCs) are a particular type of stem cells that are obtained from the inner cell mass (ICM) of the preimplantation embryo and are capable of differentiating into all cell lineages derived from the three germ layers; ESCs are pluripotent. This property makes ESCs a formidable tool to: 1) study embryonic development (Spagnoli and Hemmati-Brivanlou, 2006), 2) obtain genetically

modified animals (Robertson et al., 1986; Thompson et al., 1989), 3) establish in vitro models for

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genetic diseases (Di Giorgio et al., 2007) and 4) develop new regenerative therapies in the field of biomedicine (Keller, 2005). However, the ethical and biological caveats derived from the use of ESCs in either cell replacement therapies or basic biomedical research have made the

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development of induced pluripotent stem cells (iPSCs) one of the most important advances in the

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field of biology in the last two decades (Takahashi and Yamanaka, 2016).

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Cellular reprogramming is the conversion of a terminally differentiated somatic cell to a pluripotent state similar to that of ESCs (Takahashi and Yamanaka, 2006). Somatic cells can be

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reprogrammed to iPSCs by ectopic expression of Oct4, Sox2, Klf4 and c-Myc (OSKM hereinafter) (Takahashi and Yamanaka, 2006); chemical treatment (Hou et al., 2013); or somatic nuclear

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transfer (Cibelli et al., 1998; Munsie et al., 2000; Wakayama et al., 2001). Among the different

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approaches, OSKM-induced somatic cell reprogramming has become the most widespread

technique due to its high reproducibility, applicability to human samples and simplicity of the process. Given the important differences between the two states, this transformation entails a deep reorganization of the somatic cellular phenotype at all levels. For this dramatic phenotypic transformation to be successful, an organized sequence of events is necessary (Figure 1). This begins with the silencing of the somatic gene expression program (Stadtfeld et al., 2008). Then, an activation of the cell cycle (Mikkelsen et al., 2008), an intense mitochondrial and metabolic

remodeling (Folmes et al., 2011; Panopoulos et al., 2012; Prieto et al., 2016b; Son et al., 2015) and a mesenchymal to epithelial transition (MET) (Li et al., 2010; Samavarchi-Tehrani et al., 2010) follows. The cell conversion ends with the process of cellular immortalization (Krizhanovsky and Lowe, 2009) and the activation of early (such as SSEA1 or alkaline phosphatase) and late (such 2

Journal Pre-proof as Oct4 or Nanog) pluripotency markers (Brambrink et al., 2008; Stadtfeld et al., 2008). During this last stage of the process, cells silence the expression vectors encoding the exogenous factors used for cell reprogramming and erase all the somatic epigenetic marks from their genome (Ang et al., 2011; Ding et al., 2014; Wang et al., 2011). Both passive and active DNA demethylation mechanisms have been proposed to control this epigenetic change (Apostolou and Hochedlinger, 2013; Hochedlinger and Plath, 2009; Maherali et al., 2007; Mikkelsen et al., 2008; Polo et al., 2010). Three seminal studies have shown that cell reprogramming is organized in two waves or cascades, of cellular processes (Buganim et al., 2012; Hansson et al., 2012; Polo et al., 2012). A

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first wave is associated, fundamentally, with changes in cell cycle, metabolism and cytoarchitecture. The second wave that follows eventually leads to reactivation of the endogenous core pluripotency network, which controls pluripotency independently of the exogenous factors

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used for cell reprogramming (Figure 1). Although the epigenetic changes are continuous along all

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the stages of the reprogramming process, the reconfiguration of the DNA methylation patterns

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mainly takes place in the second phase of the process (Apostolou and Hochedlinger, 2013; Polo et al., 2012). These studies have revealed that the low efficiency of the process is due to the fact that

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numerous cells are refractory to cell reprogramming. These refractory cells are trapped in cellular

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intermediaries along the process (Figure 1). Self-renewal is the ability of stem cells to give rise to exact copies of themselves. Both ESCs

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and iPSCs undergo symmetrical self-renewal in vitro (Smith, 2009). As self-renewal is intimately linked to cell proliferation, ESCs and iPSCs must have a robust control of these two processes to preserve pluripotency division after division. Mitochondria are key organelles for cellular homeostasis: energy production, generation of intermediate anabolic metabolites, buffering intracellular calcium concentrations (Jacobson and Duchen, 2004), cell signaling, iron-sulfur protein assembly (Stehling et al., 2014), apoptosis (Tait and Green, 2010) or innate immunity (Cloonan and Choi, 2013). In this review, we will show how the processes of mitochondrial dynamics (in terms of the fission-fusion and biogenesis-degradation of these organelles) and mitochondria-regulated metabolic pathways play key roles in the acquisition as well as the maintenance of pluripotency in both ESCs and iPSCs. 3

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Figure 1. Cell reprogramming is organized in two waves or cascades of cellular processes. A first wave

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associated with changes in cell cycle, metabolism and cytoarchitecture. The second wave leads to reactivation of the endogenous core pluripotency network, which controls pluripotency independently of the exogenous factors used for cell

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reprogramming. Although the epigenetic changes are continuous along all the stages of the reprogramming process, the reconfiguration of the DNA methylation patterns mainly takes place in the second phase of the process. Numerous cells are refractory to cell reprogramming, being trapped in cellular intermediaries along the process. 5mC, 5-methylcytosine;

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5hmC, 5-hydroxymethylcytosine.

Mitochondrial dynamics during embryonic development: ESCs Mitochondria should not be considered autonomous and static, but rather as a dynamic network of organelles that act cooperating with each other in a coordinated manner. There is no de novo synthesis of mitochondria, but they divide by fission and join by fusion (Bereiter-Hahn and Voth, 1994; Johnson et al., 1981; Rizzuto et al., 1996). The balance between fission and fusion allows the mitochondria to adopt different structures (Figure 2). When fission is greater than fusion, equilibrium shifts towards fragmented and isolated mitochondria. When fusion is greater than fission, the mitochondria lengthen and acquire a tubular morphology, becoming an extensive network of interconnected mitochondria. This complex fusion and fission dynamics allows to 4

Journal Pre-proof maintain a homogeneous and functional mitochondrial population, exchanging components (proteins or copies of mitochondrial DNA, mtDNA) and/or restoring the proton gradient (Chen et al., 2003; Chen et al., 2010; Legros et al., 2002). The control of mitochondrial dynamics is therefore crucial for the correct implementation of mitochondrial functions. Mutations or deficiencies in the components that guide and regulate this dynamics generate a heterogeneous mitochondrial population and a reduction of the mitochondrial energy capacity (Chen et al., 2005; Chen et al., 2007) that are either associated to human pathologies, such as dominant optic atrophy (Alexander et al., 2000; Delettre et al., 2000) or Charcot-Marie-Tooth disease (Baxter et al., 2002; Nelis et al.,

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2002; Zuchner et al., 2004) or to neonatal mortality in mice (Ishihara et al., 2009; Wakabayashi et al., 2009). ESCs self-renewal and differentiation are opposing processes. There is a balance

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whereby pluripotent cells either differentiate or continue to proliferate and self-renew. Imbalances in mitochondrial dynamics break that equilibrium and greatly affect self-renewal and differentiation

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of PSCs.

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Mitochondria follow a maternal inheritance as it is the oocyte which supply the zygote with these organelles during fertilization. In human female oogenesis mitochondria are characterized by

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being a population of rounded organelles (<1 µm) with small vesicular cristae (folds of internal

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mitochondrial membrane). During oocyte maturation matrix become denser, cristae enlarge but rarely traverse the matrix and mitochondrial number increases, becoming the most outstanding

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organelle of the oocyte (Motta et al., 2000). An increment from approximately 10,000 mitochondria in primordial oocytes to around 100,000 in mature oocytes has been described (Jansen and de Boer, 1998). After fertilization mitochondria suffer a morphological evolution from oval-shaped mitochondria with an electro-dense matrix and few arched cristae to a more elongated forms of the organelle (1.5-2.5 µm) with a lighter matrix and more numerous cristae transversally disposed to the long axis of the mitochondria. However, a heterogeneous mitochondrial population co-exists during the subsequent cleavage stages: ICM and trophectoderm contain spherical and elongated mitochondria, respectively (Motta et al., 2000; Sathananthan and Trounson, 2000; Van Blerkom, 2009). During the first phases of embryonic development mitochondrial mass remains relatively constant. Homozygous mutant embryos for mitochondrial transcriptional factor A (Tfam), a 5

Journal Pre-proof mitochondrial transcription factor necessary for mtDNA replication, proceed to implantation and gastrulation, but die prior to embryonic day 10.5, suggesting that neither mitochondrial biogenesis nor mtDNA replication are activated during these stages (Larsson et al., 1998). Consequently, it has been described that mature oocytes before fertilization and egg-cylinder-stage embryos contain similar levels of mtDNA, approximately 100,000 copies per embryos (Ebert et al., 1988; Piko and Matsumoto, 1976; Piko and Taylor, 1987). Thus, the existing mitochondria are distributed between daughter cells in each cell division and the original mitochondrial load from oocyte must be sufficient to meet the energy requirements of the blastomeres during successive cell divisions.

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As infertile oocytes contain significantly fewer copies of mtDNA (Reynier et al., 2001; Santos et al., 2006; Wai et al., 2010), a minimum number of mitochondria necessary for proper fertilization and

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embryonic development has been proposed. The mtDNA content in mature mouse and human oocytes varies from 10,000 to 650,000 copies or from 20,000 to 800,000 copies, respectively, and

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starts to increase after implantation (Harvey, 2019; Van Blerkom, 2009). Wai and colleagues

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described that oocytes with as few as 4,000 copies of mtDNA can be fertilized and develop through preimplantation stages whereas a threshold of 40,000 to 50,000 mtDNA copies in a

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mature mouse oocyte were proposed to be sufficient for proceeding to post-implantation development (Wai et al., 2010). MtDNA copy number has been used as a good proxy of

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mitochondrial content. Some estimations have assumed one or two copies of mtDNA per

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mitochondrion (Cummins, 2002; Jansen, 2000) for establishing a minimum number of mitochondria (May-Panloup et al., 2007; Van Blerkom, 2011). Those values may have important clinical implications for fertilization and developmental competence; however, several conditions can affect the number of nucleoids per organelle, such as dynamics, morphology and species (Gilkerson et al., 2013; Yan et al., 2019), so it remains unknown the real utility of such thresholds. Although it is well established that mitochondria follow a maternal inheritance, the presence of paternal mitochondria has been shown after fertilization in mammals (Sathananthan and Trounson, 2000). Recent studies have described that fertilization triggers selective autophagy and ubiquitin/proteasome system-dependent elimination of paternal mitochondria and prevent the transmission of paternal mtDNA to progeny (Al Rawi et al., 2011; Song et al., 2016). In mammals, 6

Journal Pre-proof such as rhesus monkey and pig, the paternal mitochondria are modified with ubiquitin after fertilization, and then selectively eliminated by the proteasome or lysosome (Song et al., 2016). In fact, autophagy is essential for preimplantation development. The canonical autophagy pathway is activated in fertilized rat oocytes 4 hours after fertilization and fertilized mouse oocytes lacking this pathway do not proceed beyond the 4- to 8-cell stage (Tsukamoto et al., 2008). However, the precise mechanism whereby zygote degrades paternal mtDNA remains unknown (Yan et al., 2019). Future research will be necessary to determine which specific footprint of paternal

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mitochondria triggers this selective degradation process.

Figure 2. Mitochondrial dynamics and post-translational modifications. The molecular machinery that controls fission and fusion processes includes integral mitochondrial membrane proteins and proteinic factors that are recruited to the surface of these organelles in response to wide range of stimuli. Mfn1/2 are ubiquitin-marked for degradation by Parkin (Chan et al., 2011; Tanaka et al., 2010). Erk1/2 (Pyakurel et al., 2015) and Jnk (Leboucher et al., 2012) impair mitochondrial fusion through phosphorylation of Mfn1/2. Opa1 is regulated by proteolytic processing (Griparic et al., 2007; Ishihara et al., 2006; Song et al., 2007). Drp1 is regulated by multiple post-translational modifications (Otera et al., 2013): mainly, phosphorylation of Ser579 in mouse (Ser616 in humans) by Cdk1 (Taguchi et al., 2007), Erk1/2 (Kashatus et al., 2015; Serasinghe et al., 2015), Pkc (Qi et al., 2011) and Cdk5 (Xie et al., 2015) and

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Journal Pre-proof phosphorylation of Ser656 (Ser693 in humans) by Gsk3β (Chou et al., 2012) induces mitochondrial fission. Conversely, phosphorylation of Ser600 (Ser637 in humans) by Pka (Chang and Blackstone, 2007; Cribbs and Strack, 2007; Gomes et al., 2011), Ampk (Wikstrom et al., 2013) and CamkII (Xie et al., 2015) induces mitochondrial fusion, and its dephosphorylation by calcineurin (CaN) induces mitochondrial fission (Cereghetti et al., 2008; Cribbs and Strack, 2007). In some circumstances, however, phosphorylation of Ser600 by CamkI (Han et al., 2008) or RockI (Wang et al., 2012) can induce mitochondrial fission. Ampk also favors mitochondrial fission through Mff phosphorylation in response to stress (Toyama et al., 2016).

Due to their early embryonic origin, ESCs tend to have a simple mitochondrial network: fragmented morphology, cristae-poor internal matrix and a low number of mtDNA copies.

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Conversely, somatic cells present a more complex structure of their mitochondrial network: tubular and/or interconnected morphology, well-developed internal crista and a dense mitochondrial matrix

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with a high number of mtDNA copies (Cho et al., 2006; Chung et al., 2007; Prieto et al., 2016b; Prigione et al., 2010; St John et al., 2005; Suhr et al., 2010). The low mtDNA replication rate in

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mouse ESCs appears to be related to the silencing of the genes encoding the mitochondrial DNA

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polymerase catalytic subunit γ (Polg) and Tfam by DNA methylation (Kelly et al., 2012; Larsson et al., 1998). During cell differentiation, mitochondrial mass rises, mitochondria size enlarges and the

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internal complexity of these organelles matures (Cho et al., 2006; Facucho-Oliveira et al., 2007; Spikings et al., 2007). In parallel, mtDNA replication increases because DNA demethylation of

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Tfam and Polg activates their expression after embryo implantation (Kelly et al., 2012; Larsson et

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al., 1998). For example, specification of murine ESCs and embryos towards cardiomyocytes (Chung et al., 2007; Hom et al., 2011) and human mesenchymal stem cells towards adipocytes (Tormos et al., 2011) is characterized by an increase in elongation, matrix complexity and functionality of mitochondria. Actually, chemical induction of mitochondrial fusion by hydrazone M1, a small molecule that promotes fusion of this organelle, commit human iPSCs to cardiac differentiation (Lees et al., 2019b). In addition, during cardiomyocyte specification in mouse embryonic development, closure of the mitochondrial permeability transition pore increases mitochondrial membrane potential (MMP) and reduces the intracellular levels of reactive oxygen species (ROS) (Hom et al., 2011).

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Journal Pre-proof The molecular machinery that controls fission and fusion processes includes integral mitochondrial membrane proteins and proteinic factors that are recruited to the surface of these organelles in response to a wide range of stimuli (Figure 2). The fusion of outer (OMM) and inner (IMM) mitochondrial membranes is a temporally coordinated multi-step process that is regulated by transmembrane adapter proteins Mitofusins (Mfn) 1 and 2 and optic atrophy protein 1 (Opa1) (Figure 2). Mfn1/2 and Opa1 span both membranes (Hoppins and Nunnari, 2009). Cells that lack Mfn1 or Mfn2 show fragmented mitochondria and fail in mitochondrial complementation (Chen et al., 2003; Detmer and Chan, 2007), which leads to an accumulation of dysfunctional mitochondria

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(Chen et al., 2005). In the absence of Opa1, both OMMs merge but do not share the components of the mitochondrial matrix due to the lack of inner mitochondrial membrane fusion (Meeusen et

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al., 2006). Mfn2 and Opa1 proteins play a key role in mitochondrial maturation during cell differentiation. The absence of Mfn2 or Opa1 prevents mitochondrial fusion, which leads to an

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increase in cytosolic calcium levels and the activation of calcineurin, eventually impairing the

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efficient differentiation of mouse ESCs into cardiomyocytes (Kasahara et al., 2013). In contrast, the pharmacological inhibition of dynamin-related protein 1 (Drp1), the main protein responsible of

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mitochondrial fission (Otsuga et al., 1998; Smirnova et al., 1998) in combination with adapter proteins and the endoplasmic reticulum (ER) (Friedman et al., 2011; Korobova et al., 2013)

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(Figure 2), reduces mitochondrial fission, favors oxidative phosphorylation and therefore facilitates

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the specification of human PSCs towards cardiac mesoderm (Hoque et al., 2018). Drp1-null mouse ESCs have been derived by homologous recombination. Although Drp1 knock-out mice show significant defects in embryonic development and synapse formation (Ishihara et al., 2009; Manczak et al., 2012; Wakabayashi et al., 2009), murine Drp1-null ESCs maintain pluripotency and the capacity to self-renew (Ishihara et al., 2009). Drp1 knock-out cells show a tubular mitochondrial morphology and a lower proliferation rate. Surprisingly, the lack of Drp1 gene does not affect cytokinesis. Given the central role of Drp1 in mitochondrial fission and that this process

is critical to ensure an proper distribution of these organelles between the two daughter cells upon cell division (Katajisto et al., 2015; Taguchi et al., 2007), the results obtained by Ishihara and colleagues were puzzling. To explain this paradox, the authors suggested that unknown mechanical forces could play a role in the segregation of mitochondria between the two daughter 9

Journal Pre-proof cells during cell division. Whether Drp1-null somatic cells could be reprogrammed to pluripotency remains unexplored. An inadequate fusion-fission balance can compromise the proper maintenance of a homogeneous and functional mitochondrial population. Conversely to the conventional idea that mitochondrial fission is a mechanism to guarantee the even distribution of mitochondria in daughter cells during cytokinesis, recent work have shown that mitochondrial fission also drives

the asymmetric distribution of these organelles during cell division of human mammary stem-like

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cells (Katajisto et al., 2015), which adds an additional layer of complexity to our classical understanding of the functional role of mitochondrial fission in cell division. This asymmetric distribution of mitochondria depends on the quality of the organelles, and aged or deficient

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organelles were segregated to the most differentiated daughter cell whereas healthy mitochondria

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were allocated to the remaining daughter cell during cytokinesis. Interestingly, the daughter cells

that retained the most healthy and functional mitochondria had stem cell characteristics. It is

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thought that this asymmetry in the segregation of mitochondria contributes to maintain a homogeneous and healthy population of stem cells, which could be considered as a kind of selfish

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self-renewal. In addition, it has been described that overexpression of Drp1 in mouse ESCs

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diminishes expression of pluripotency markers, impairs embryoid body formation and induces apoptosis (Todd et al., 2010). In mouse neural stem cells (NSCs), mitochondrial dynamics is

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apparently displaced towards mitochondrial fusion rather than fission, where Mfn2 and Opa1 play a fundamental role in their self-renewal (Khacho et al., 2016). This aberrant mitochondrial dynamics in the absence of Mfn2 and Opa1 impairs stem cell self-renewal and maintenance, inducing stem cell exhaustion and differentiation. NSCs suffer an archetypical switch in metabolism during cell differentiation: whereas Sox2-positive uncommitted cells are mainly glycolytic, the transition of NSCs to a committed fate is paralleled by an induction of a metabolic switch to oxidative phosphorylation (OXPHOS). Increased OXPHOS raises ROS levels and activates the differentiation program. However, this cell conversion seems to follow an opposite transition in mitochondrial dynamics: Sox2-positive uncommitted cells contain elongated mitochondria and suffer a drastic shortening of mitochondrial length during cellular commitment. In addition, Mfn2 10

Journal Pre-proof also participates in the proper self-renewal of hematopoietic stem cells (HSCs) in mouse. In these cells, Mfn2 participates in maintaining an adequate level of intracellular calcium by ensuring ERmitochondrial contacts (Luchsinger et al., 2016). Together, these data suggest that mitochondrial dynamics typically shifts to the fission of these organelles during stem cell self-renewal. Besides mitochondrial dynamics, other cellular features (such as their proliferation rate, association with their niche or cell fate upon cellular division) may differentially influence the mitochondrial fusionfission balance, which could also be cell type-specific.

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Mitochondrial dynamics during cell reprogramming: iPSCs Different processes of cell reprogramming have been developed: somatic cell nuclear transfer (SCNT), cell fusion, ectopic OSKM expression or chemical compound exposure. Each

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strategy display different features: timing, epigenetic remodeling and somatic-to-pluripotency

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developmental routes (Cacchiarelli et al., 2015; Takahashi et al., 2014; Velychko et al., 2019;

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Zhao et al., 2015). However, cell reprogramming and cell differentiation always have opposite developmental-start and -end points. In this regard and conversely to the tubular mitochondrial

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network observed in most somatic cells, iPSCs and ESCs, have a cristae-poor and fragmented mitochondrial morphology (Folmes et al., 2011; Prieto et al., 2016b; Prigione et al., 2010; Vazquez-

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Martin et al., 2016). During this cellular conversion mitochondria become healthier and gain

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functionality: MMP increases (Prieto et al., 2018), ROS-induced senescence is repressed (Prigione

et al., 2010), OXPHOS capacity is restored (Kida et al., 2015; Prieto et al., 2018), mtDNA mutation frequency decreases (Prigione et al., 2011) and mitochondria marked for degradation is reduced (Wang et al., 2019). Interestingly, since in vivo cell reprogramming was described (Abad et al., 2013), in vivo partial cell reprogramming has been developed and proposed as an anti-aging strategy (Ocampo et al., 2016a; Ocampo et al., 2016b).The role of mitochondria in aging is well known (Zhang et al., 2018). Together, these data allows us to conclude that cell reprogramming

induces a mitochondrial rejuvenation and provides a new insight for future mitochondrial strategies against aging (Madreiter-Sokolowski et al., 2018). The reorganization of the mitochondrial network is one of the first cellular barriers that somatic cells face during cell reprogramming (Figure 3). The cellular intermediaries and the cells 11

Journal Pre-proof of the epithelial colonies that appear during the first days of the process from mouse embryonic fibroblasts (MEFs) have a fragmented mitochondrial morphology, similar to that observed in ESCs, suggesting that mitochondrial reorganization occurs during early stages (Prieto et al., 2016b; Son et al., 2015). Mouse iPSCs and ESCs have high Drp1 levels (Prieto et al., 2016b). Although this protein has been described as dispensable for cell reprogramming in mouse (Wang et al., 2014), multiple studies have shown that chemical (Vazquez-Martin et al., 2012a) or functional (Prieto et al., 2016b) inhibition and gene knockdown (Prieto et al., 2016b; Son et al., 2013b) of Drp1 prevents cell reprogramming, whereas its activation by phosphorylation favors the reprogramming

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process (Prieto et al., 2016b; Son et al., 2015; Son et al., 2013b). Drp1 is a cytosolic protein with GTPase activity that is activated by post-translational modifications in response to different stimuli and recruited to the mitochondrial surface through interaction with protein adapters (van der Bliek

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and Payne, 2010) (Figure 2). During the early phases of cell reprogramming, there is an increase

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in both total protein and phosphorylation of the Ser579 residue of Drp1 in MEFs (Figure 3). During

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this early phase, Erk1/2 are activated as a the result of the negative regulation of Dusp6 MAPkinase phosphatase by OSKM. Activated Erk1/2 then phosphorylates Drp1-S579, which induces

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Drp1 recruitment to mitochondria and triggers mitochondrial fission (Prieto et al., 2016b). In reprogramming

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proliferation,

favoring

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phosphorylation of Drp1 through the activation of cyclin-dependent kinase 1 (Cdk1) by increasing

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Cyclin B protein levels (Prieto et al., 2018). Accordingly, it has been observed that the transcription factor REX1 activates the expression of CYCLIN B in human ESCs. This increment of CYCLIN B levels activates the CDK1/CYCLIN B complex, which leads to an increase in phosphorylation of Drp1-S579 and mitochondrial fragmentation. In agreement with these results, REX1-null human ESCs show a tubular mitochondrial morphology and decreased self-renewal capacity (Son et al., 2013b). Compared to somatic cells, mouse ESCs and cell reprogramming intermediaries show low levels of Mfn1/2 expression (Prieto et al., 2016b; Son et al., 2015). Interestingly, chemical inhibition or knockdown of Mfn1/2 led to a faster and higher efficiency of cell reprogramming due to an increase in mitochondrial fragmentation and cell proliferation. At the same time, lack of Mfn1/2 12

Journal Pre-proof favored the activation of Erk1/2, which may facilitate the phosphorylation of Drp1-S579 by these MAP kinases (Son et al., 2015). In addition, Erk1/2-mediated phosphorylation of Mfn1 causes its inactivation in MEFs (Pyakurel et al., 2015) (Figure 2). Therefore, in addition to increasing mitochondrial fission through phosphorylation of Drp1 (Prieto et al., 2016b), activation of Erk1/2 early in cell reprogramming may inhibit mitochondrial fusion through phosphorylation of Mfn1 (Figure 3).

Different protein adapters for Drp1 have been described, including mitochondrial fission

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protein 1 (Fis1) (Mozdy et al., 2000), mitochondrial fission factor (Mff) (Otera et al., 2010) or mitochondrial dynamic proteins of 49 kDa (Mid49) and 51 kDa (Mid51) (Palmer et al., 2011; Zhao et al., 2011) (Figure 2). However, it seems that only Mid51 is involved in mitochondrial fission

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during cell reprogramming. Reduction of Mid51 protein levels reduced the efficiency of cell

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reprogramming in MEFs (Figure 3) (Prieto et al., 2016a). It remains unknown whether ER has any

active role during cell reprogramming. Interestingly, we described that knockout cells for

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ganglioside-induced differentiation associated protein 1 (Gdap1) showed a lower reprogramming efficiency due to a defect in the activation of mitochondrial fragmentation during the process

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(Figure 3). The inability of Gdap1-null cells to undergo efficient mitochondrial fission during cell

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reprogramming induced a G2/M arrest in a DNA damage-independent manner (Prieto et al., 2016a). A pro-fission role for Gdap1 has been proposed, as Gdap1 favors the formation of ER-

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mitochondria contacts in certain types of neural cells (Pla-Martin et al., 2013) and its overexpression leads to fragmented mitochondria (Lopez Del Amo et al., 2015; Niemann et al., 2005). Therefore, it is not unreasonable to hypothesize that lack of Gdap1 may reduce the number of ER-mitochondria contacts during cell reprogramming, reducing the ability of the cells to fragment these organelles and to progress towards the iPSC state. Beyond the role of ER in mitochondrial fission during cell reprogramming, it has recently been described that unfolded protein response (UPR) of ER and mitochondria are activated during cell reprogramming. The UPR of endoplasmic reticulum is particularly crucial, and its ectopic transient activation enhances cell reprogramming (Simic et al., 2019). These pathways guarantee proteostasis during stress and aging (Brehme et al., 2014), highlighting the potential of cell reprogramming as an anti-aging therapy. 13

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Figure 3. Reorganization of the mitochondrial network is one of the first cellular barriers that somatic

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cells face during cell reprogramming. The cellular intermediaries and the cells of the epithelial colonies early in reprogramming have a fragmented mitochondrial morphology, similar to that observed in the ESCs. Compared to

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somatic cells, mouse ESCs and cellular intermediaries show low and high levels of Mfn1/2 and Drp1 expression, respectively. Phosphorylation of Drp1 by Cdk1 and Erk1/2 drives mitochondrial fragmentation early in cell

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reprogramming, while Mfn1/2 phosphorylation impairs the process and reduce cell reprogramming efficiency. Activation of Erk1/2 early in cell reprogramming may inhibit mitochondrial fusion through phosphorylation of Mfn1. Mid51 and

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Gdap1 are also involved in mitochondrial fission during cell reprogramming. Gdap1 may reduce the number of ERmitochondria contacts during cell reprogramming, reducing the ability of the cells to fragment these organelles and

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therefore progress towards the iPSC state.

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Compared to the somatic state, mouse and human iPSCs show a reduction in the complexity and number of mitochondria (Folmes et al., 2011; Prigione et al., 2010; Suhr et al., 2010). Mitochondrial fission is part of the process regulating mitochondrial mass and function through mitophagy (Ashrafi and Schwarz, 2013). As a mitochondrial clearance during cell reprogramming (Prigione and Adjaye, 2010; Prigione et al., 2010; Wang et al., 2013) and a protective role of autophagy in the maintenance and self-renewal of mouse ESCs (Liu et al., 2017), HSCs (Ho et al., 2017; Warr et al., 2013) and muscle stem cells (MuSCs) (Garcia-Prat et al., 2016) have been described, some studies have suggested that mitophagy may be involved in the reduction of mitochondrial mass and therefore play a positive role in the process of acquisition and maintenance of pluripotency (Pan et al., 2013; Vessoni et al., 2012). Accordingly, autophagy inductors (such as rapamycin, spermidine or AICAR) have been described to increase the 14

Journal Pre-proof efficiency of cell reprogramming (Chen et al., 2011; Ma et al., 2015) and an early and transient activation of this process has been observed during the reprograming process (Liu et al., 2016; Wang et al., 2013; Xiang et al., 2017). However, new studies have cast doubt on these results. The work carried out by three different laboratories not only showed that Lc3b/Atg5-dependent autophagy is not responsible for the mitochondrial clearance observed during cell reprogramming (Ma et al., 2015; Prieto et al., 2016b; Wang et al., 2019), but also that its early activation supposes a brake on the process of cellular reconversion (Wu et al., 2015). Given the high cellular heterogeneity that exists during the first days of the process, this paradox may be due to multiple

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causes: the reprogramming model used (transcriptional factor combination, vectors, genetic approach, etc.), the culture medium used (which determines the efficiency of the reprogramming

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process), the basal autophagy levels present in the cells subjected to reprogramming, total number of cells analyzed to power statistical significance or methods used for assessing mitophagy.

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Recently, Wang and cols. have deeply looked into available bibliography in this regard and

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provided new evidences to conclude that mitophagy is not necessary for mitochondrial clearance (Wang et al., 2019). The authors demonstrate that Ulk1-mediated autophagy is unnecessary for

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reprogramming and that mitochondrial remodeling is dependent on the inhibition of mitochondrial

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biogenesis through the mTorc1-Pgc1 pathway. Therefore, it seems unlikely that mitophagy plays an active role in the mitochondrial

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clearance during cell reprogramming. These observations suggest that, beyond the role of basal renewal of dysfunctional mitochondria, mitophagy is not necessary for cell reprogramming. It is possible that the absolute reduction of the mitochondrial mass is due to a passive process of dilution cycle after cycle, an adaptation to the new cell culture conditions required to maintain pluripotency (Prieto et al., 2016b; Xiang et al., 2017) and/or a decrease in mitochondrial biogenesis (Wang et al., 2019). It is also worth mentioning that as mitochondrial network is remodeled during cell reprogramming, cells are downsized: mitochondrial mass decreases whereas cells become smaller (Prieto et al., 2016b; Prieto et al., 2018; Wang et al., 2019). In addition, observations from two additional laboratories showed that human ESCs have a proportion of mitochondrial mass/total protein similar to somatic cells (Birket et al., 2011; Zhang et al., 2011). Thus, it does not escape to 15

Journal Pre-proof our notice that such reduction of mitochondrial mass may be a simple effect of cell size alteration. In this regard, it would be interesting to investigate whether molecular crowding (Richter et al., 2008) triggered during cell reprogramming plays a role during the process.

Metabolic dynamics during embryonic development

Cellular metabolism is tightly linked to mitochondria. Dynamics, mass, mtDNA numbers and mitochondrial functionality affects the way cells obtain energy. During first stages of embryonic

metabolism and mitochondrial dynamics takes place.

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development, ranging from zygote to gastrulation stage, a profound reorganization of cellular

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During the states of oocyte and fertilized egg, energy is primarily obtained by oxidation of

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pyruvate and lactate, rather than glucose (Brinster and Troike, 1979; Downs et al., 2002; Leese

and Barton, 1984; Martin and Leese, 1995) (Figure 4). In fact, high amounts of these substrates

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have been detected in the reproductive tract fluid (Brinster, 1965; Gardner et al., 1996) secreted by

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cumulus cells, a defined group of cells that surround the oocyte and participate in the processes of oocyte maturation and fertilization. They actively produce pyruvate and lactate from glucose

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(Leese and Barton, 1985). The first two days of development are characterized by low metabolic activity because there is no significant increase in biomass and a distribution of the original

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components of the zygote takes place upon cell division. In fact, a high ATP/ADP ratio exists before embryo compactation, driving the embryo independence of glucose uptake (Leese et al., 1984). Also, a high ratio of ATP/ADP inhibits phosphofructokinase (PFK) allosterically, impairing glycolysis flux (Figure 5) (Barbehenn et al., 1974). From this point onwards, compactation starts and energy demand soars for attending cell proliferation (from a 16-cell morula to a blastocyst of 100–200 cells) and blastocoel formation.

Concomitantly, ratio of ATP/ADP decreases, glucose uptake increases and glucose starts to replace pyruvate and lactate as primary energy sources (Leese and Barton, 1984) (Figure 4). However, blastocyst still have a considerable capacity to use pyruvate (Gardner et al., 2001) and, in the absence of glucose, the blastocyst can increase pyruvate uptake to counteract this fact 16

Journal Pre-proof (Gardner and Leese, 1988). Gradually, energy production from glucose changes: firstly by oxidative phosphorylation and secondly by aerobic glycolysis (Houghton et al., 1996). This trend peaks at the time of implantation, where aerobic glycolysis is the main route for energy production in the ICM (Gardner and Leese, 1990) (Figure 4). The expression of glucose transporters and glycolytic enzymes increases at this point, and their mutation is lethal at the blastocyst stage (Jansen et al., 2008; Pantaleon and Kaye, 1998; Shyh-Chang et al., 2013). Interestingly, once trophectoderm appears, it maintains an oxidative metabolism during all the stages (Hewitson and Leese, 1993), corresponding to more elongated mitochondria with large cristae that traverse the

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matrix (Mohr and Trounson, 1982). This elevated oxygen consumption in trophectoderm is necessary for blastocoel formation due to a high ATP demand of Na+/K+-ATPase for sodium ion

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pumping into extracellular space (Watson and Kidder, 1988). The osmotic gradient created elicits the water movement inside the blastocyst, facilitated by the presence of aquaporins (Barcroft et al.,

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2003). It has been suggested that dependence on glycolysis at this point may be due to insufficient

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presence of oxygen: oxygen concentration in the lumen of the female reproductive tract is 2%-8% (Fischer and Bavister, 1993; Mitchell and Yochim, 1968) and the embryo does not contact with

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maternal blood oxygen until placenta is formed after implantation (from second week to third month in humans) (Sherer and Abulafia, 2001). Interestingly, culture of preimplantation embryos under

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hypoxic conditions (typically around 5%) elicits higher rates of implantation and development in

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several mammalian species whereas normoxia culture conditions (20%) affects transcriptome, proteome and metabolism of embryos during the preimplantation period (Gardner and Harvey, 2015; Harvey, 2007). These particular physiological conditions together with knockout studies highlight the relevance of hypoxia inducible factors (HIFs) during the early stages of embryonic development (Gardner and Harvey, 2015). It has been described that key glycolytic genes, such as glucose transporter 1 (Glut1) and lactate dehydrogenase (Ldh), are significantly increased in bovine blastocysts in 2% oxygen culture conditions in the presence of Hif2 (Harvey et al., 2004a).

In this regard, development of bovine embryos to the blastocyst stage is improved in the presence of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation which increases mRNA of Hif2 (Harvey et al., 2004b). However, low concentration of oxygen (2%) increase oxygen-regulated gene expression above levels found in normoxia-cultured or in vivo-derived mouse embryos (Kind 17

Journal Pre-proof et al., 2005). Also, mouse embryos cultured under hypoxic conditions show lower rates of posttransfer development (Feil et al., 2006). Species-specific responses to reduced oxygen concentration may reflect differences in the developmental process (implantation, gastrulation, heterochrony). Beyond these particular physiological conditions during early stages of embryonic development, a second commitment for the glycolytic phenotype has been proposed. Remarkably. David Gardner lab identified metabolic similarities between cancers and blastocysts (Gardner, 1998) and thus, the impact of the Warburg effect in these stages of development has been widely reviewed (see below and reviewed by Krisher and Prather, 2012; Redel et al., 2012; Smith and

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Sturmey, 2013). Furthermore, D. Gardner lab has suggested that aerobic glycolysis creates a very especial microenvironment around the embryo, characterized by high lactate and low pH, features

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that may facilitate uterine invasion and implantation. A high lactate/low pH microenvironment created by the blastocyst may help to degrade i) extracellular matrix, ii) induce angiogenesis and

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iii) modulate the activity of the local immune response, therefore favoring immune tolerance

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(Gardner, 2015).

Before implantation, the energy demand is relatively low within the cells. This low amount of

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ATP correlates with a still reduced mitochondrial mass and a low copy number of mtDNA (see

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above). However, as the embryos advance in its development, energy requirements become higher, specially after implantation, when gastrulation ensues and the single-layered blastula is

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reorganized into a multilayered (ectoderm, mesoderm and endoderm) structure known as gastrula. Gastrulation is followed by organogenesis and a profound process of cell differentiation starts. Cell differentiation requires an extreme process of epigenetic remodeling to undergo cell specification, which greatly increases the demands of energy as well as other mitochondria-derived metabolites, such as acetyl-CoA, NADH, s-adenosylmethionine (SAM) or α-ketoglutarate (see below and reviewed by Ryall et al., 2015). For this reason, a suitable embryo development requires a reliable mitochondrial function to guarantee a proper cell differentiation during organogenesis in later stages. Regarding this point, alterations in mitochondrial activity have been found to impact fertilization, implantation and developmental potential. There is a correlation between ATP content in human oocytes and development after in vitro fertilization and embryo transfer (Van Blerkom et 18

Journal Pre-proof al., 1995). Also, proper ATP levels have been shown to guarantee normal meiotic and mitotic spindle organization and chromosomal segregation during meiosis in the oocyte and early cleavage in the embryo, respectively (Schon et al., 2000; Zeng et al., 2007). In this regard, it has been shown that mitochondria with high MMP is necessary for fertilization (Van Blerkom and Davis, 2007), cytokinesis during cleavage (Van Blerkom and Davis, 2006) and apoptotic cell death

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during the preimplantation stage (Acton et al., 2004).

Figure 4. Cellular metabolism is tightly linked to mitochondria during embryonic development. During the

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state of oocyte and fertilized egg, energy is primarily obtained by oxidation of pyruvate and lactate rather than glucose. From this point onwards, compactation starts and energy demand soars for attending cell proliferation and blastocoel

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formation. Glucose uptake increases and glucose starts to replace pyruvate and lactate as primary energy sources. Gradually, the production of energy from glucose changes: firstly, by oxidative phosphorylation and secondly, by aerobic glycolysis. This trend peaks at the time of implantation, where aerobic glycolysis is the main route for energy production. OXPHOS and glycolysis rates and mitochondrial morphology of cells from ICM are indicated, trophectoderm is not shown. Once trophectoderm appears, it maintains an oxidative metabolism necessary for blastocoel formation. During organogenesis, cell differentiation requires large amounts of ATP and other mitochondria-derived metabolites. Pyr (pyruvate), Glc (glucose) and Gln (glutamine) refers to main source of carbon for providing substrates to oxidative phosphorylation. The oxidation of glutamine maintains an adequate metabolic rate in the Krebs cycle from blastocyst to implantation state, guaranteeing the production of ATP and fatty acids. Embryonic time points of naïve and primed ESCs origins are indicated (Table 1).

Mitochondrial maturation and biogenesis take place during cell differentiation (see above). In parallel, OXPHOS increases and ATP levels rise considerably (Cho et al., 2006; Lonergan et al., 19

Journal Pre-proof 2006; Mandal et al., 2011; Spikings et al., 2007) (Figure 4). For instance, during differentiation into mouse cardiomyocytes (Chung et al., 2007; Hom et al., 2011; Hoque et al., 2018), murine dopaminergic neurons (Pereira et al., 2013) or human adipocytes (Tormos et al., 2011) mitochondrial maturation parallels OXPHOS increase. The dramatic changes that mitochondrial morphology undergoes during embryonic development or in vitro differentiation have classically been associated with a process of architectural maturation of these organelles. This maturation would allow to implement efficient OXPHOS in differentiated cells, guaranteeing a sufficient energy production. In this regard, there are numerous reports that correlate a mature and tubular

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mitochondrial morphology with an increase in OXPHOS, and a fragmented and immature mitochondrial morphology with an increase in aerobic glycolysis and a decrease in oxidative

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metabolism (Baker et al., 2019; Mishra and Chan, 2016). Therefore, tubular mitochondria would guarantee 1) better maintenance of membrane potential, 2) homogeneous mtDNA population and

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3) adequate cytoarchitecture of the organelle to favor the formation of supercomplexes in the

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electron transport chain and increasing the efficiency of electronic transport. However, both early embryonic cells (Gardner and Harvey, 2015) and ESCs (Birket et al., 2011; Zhang et al., 2011;

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Zhou et al., 2012) have fragmented mitochondria with an oxidative capacity similar to somatic cells. Thus, it is possible that the existence of a estable fragmented mitochondrial population, with

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an active fission-fusion dynamics, facilitates the functionality of these organelles in ESCs.

Metabolism in proliferating cells: ESCs and iPSCs ESCs and iPSCs display a high division rate. Proliferating cells have a metabolism very different from non-proliferating cells (Newsholme, 1990). Cells with a high proliferation rate usually base their energy production on aerobic glycolysis: conversion of glucose to lactate in the presence of oxygen (Figure 5). This metabolic phenotype is known as the Warburg effect. Warburg observed that cancer cells consumed large amounts of glucose compared to somatic cells, and that it was metabolized primarily through aerobic glycolysis in the form of lactate (Warburg, 1956). Thus, it has been described that the stimulation of glycolysis in pluripotent cells by either hypoxic conditions (Ezashi et al., 2005; Lees et al., 2019a; Mohyeldin et al., 2010) or inhibition of OXPHOS 20

Journal Pre-proof (Varum et al., 2009) favors self-renewal. In fact, the central core of pluripotency control (Oct4Sox2-Nanog transcriptional axis), shares a multitude of binding loci with Stat3 (Torres and Watt, 2008), a major regulator of the metabolic switch from OXPHOS to glycolysis (Folmes et al., 2012). Cells need energy, usually in form of ATP, to guide non-spontaneous reactions and maintain cell homeostasis. In addition, proliferative cells have additional requirements for cell growth and division. They need to capture nutrients to metabolize them into biosynthetic precursors and coordinate the synthesis of macromolecules necessary for the generation of a new cell (Lunt and Vander Heiden, 2011). Glucose is the main source of energy from diet, which is transformed to

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pyruvate by the glycolytic pathway, producing two molecules of ATP during this process. Pyruvate can then enter into the mitochondrial Krebs cycle to produce a greater amount of ATP through the

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electron transport chain (34 additional molecules) or become lactate in the cytosol (Figure 5).

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Glycolysis (2 molecules of ATP per 1 molecule of glucose) is a less efficient route than

OXPHOS (36 molecules of ATP per 1 molecule of glucose). However, under conditions of high

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glucose content, it can produce ATP more quickly (Guppy et al., 1993). Historically, it was thought that the Warburg effect occurred due to the hypoxic situation in which cancer cells are found in the

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body or by mitochondrial respiration defects in these cells (Moreno-Sanchez et al., 2007). Notably,

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this seems not to be the case. It has been described that mitochondria in cells displaying a metabolic Warburg effect, such as ESCs (Birket et al., 2011; Zhang et al., 2011; Zhou et al., 2012),

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CSCs (Zu and Guppy, 2004) or primary lymphocytes (Macintyre and Rathmell, 2013) are perfectly functional. The analysis of 31 tumor cell lines revealed that the contribution of glycolysis to ATP production can range between 0.34% to 64% (Zu and Guppy, 2004). However, the production of ATP seems not to be limiting in proliferative cells. Some estimates suggest that the biosynthetic processes to produce a new cell do not consume large amounts of ATP (Kilburn et al., 1969). Cells such as ESCs or iPSCs only consume energy to proliferate, while somatic cells must perform a specific function in the body that demands energy. These specialized cellular functions, such as the synthesis of hormones or the transmission of nerve impulse, can be more complex and may require larger amounts of ATP than that of powering cell division (Lunt and Vander Heiden, 2011). Also, a lower demand of ATP may require a lower activity of OXPHOS in ESCs, reducing the 21

Journal Pre-proof levels of ROS and therefore favoring genomic stability. In this regard, ESCs express high levels of antioxidant enzymes, such as catalase (Cho et al., 2006), superoxide dismutase, glutathione peroxidase (Zhou et al., 2016) or uncoupling protein 2 (Zhang et al., 2011), and are prepared to resist the adverse effects of ROS, such as senescence induced by oxidative stress (Guo et al., 2010). Interestingly, there is a dual metabolic state in pluripotent cells (Table 1). Whereas primed

ESCs (Ying et al., 2008) are fundamentally glycolytic, naïve ESCs display a dual metabolic state

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(Zhou et al., 2012). This fact highlights the role of mitochondria in naïve ESCs and resembles embryonic development: glycolysis peaks at implantation stage, source of primed ESCs (Figure 4). This metabolic change is driven by Hif1, which reduces the expression of components of the

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electron transport chain and activates the expression of Ldh or the pyruvate dehydrogenase kinase

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(Pdk) (Zhou et al., 2012). HIFs are one of the main transcription factors that favor the conversion of

oxidative to glycolytic metabolism both in cancer (Semenza, 2012) and stem cells (Ito and Suda,

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2014). One of the key steps in this metabolic conversion is a reduction in the activity of Pdh, the enzyme that catalyzes the reaction of pyruvate to acetyl-CoA, favoring its entry into the Krebs

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cycle (Figure 5). Hif1 activates Pdk, which reduces Pdh activity in MEFs by direct phosphorylation

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(Kim et al., 2006). A correlation in the expression of HIFs and that of the components of the transcriptional core of pluripotency (Oct4, Sox2 and Nanog) has been observed (Teslaa and

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Teitell, 2015), and culture of human ESCs under hypoxic conditions (5%) increases glucose consumption and lactate production (Harvey et al., 2016b), raising levels of acetylated H3K9 and H3K27 and decreasing trimethylated H3K27 (Lees et al., 2019a), features of the euchromatic structure in naïve mouse PSCs (Carey et al., 2015; Moussaieff et al., 2015). The reason for the existence of two metabolic states in ESCs (naïve-bivalent versus primed-glycolytic) remains unclear. Historically, as in the case of cancer, it has been suggested

that activation of HIFs may indicate that the oxygen concentration is not sufficient to keep the electronic transport chain active during implantation. However, we propose an alternative explanation. Blocking mitochondrial function does not impair pluripotency of self-renewing ESCs. Nevertheless, early differentiation is affected when cell conversion is conducted under conditions 22

Journal Pre-proof that attenuate mitochondrial activity (Mandal et al., 2011). Primed iPSCs display hyperpolarized mitochondria with high MMP (Armstrong et al., 2010; Folmes et al., 2011; Prieto et al., 2018) and ESCs with high MMP have a greater capacity for teratoma formation (Schieke et al., 2008). Together these data support the notion that, even though mitochondrial function is not strictly necessary for self-renewal of ESCs, only PSCs with a functional mitochondrial network are capable to differentiate properly. Actually, a gradual increase in MMP has been described during in vivo and in vitro zygote-to-blastocyst development (Acton et al., 2004). We suggest that primed ESCs is a poised energetic state by which the mitochondrial hyperpolarization may act as an

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energy reservoir, accumulated in form of proton gradient, to produce large amounts of ATP that meet the high energy demand for epigenetic remodeling once cells engage differentiation after

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gastrulation.

Table 1. Molecular, functional and metabolic comparison of naïve and primed PSCs. Naïve and primed PSCs differ on molecular and epigenetic patterns. These differences give rise to a disparity in their developmental potential. Each state is characterized by a specific metabolic profile: naïve-bivalent state and primed-glycolytic state. Each metabolic state also results in a different dependency of glutamine. Lif, leukemia inhibitory factor; 2i, inhibitors for Mek1/2 and Gsk3β; Fgf2, fibroblasts growth factor 2; Xa, chromosome X activated; Xi, chromosome X inactivated; MMP, mitochondrial membrane potential. (1) Mouse epiblast-derived stem cell (EpiSCs) are included. (2) Cell culture conditions for murine PSCs. Human PSC culture conditions remains unstandardized (Boroviak and Nichols, 2017; Dakhore et al., 2018).

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Journal Pre-proof Biosynthetic pathways in ESCs and iPSCs ESCs not only have to maintain a high glycolytic flow for energetic reasons, glycolysis is crucial to provide intermediaries for biosynthetic pathways (Newsholme, 1990; Newsholme et al., 1985). For example, glucose-6-phosphate enters pentose phosphate pathway to ensure the biosynthesis of nucleotides, necessary for DNA replication, and provide reducing power in the form of NADPH for biosynthetic pathways. Fructose-6-phosphate and glyceraldehyde-3-phosphate can

also enter and leave the pentose phosphate pathway. In addition, dihydroxyacetone phosphate is

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crucial for biosynthesis of phospholipids and triacylglycerols, the major components of cell membranes; 3-phosphoglycerate participates in the synthesis of phospholipids and amino acids, such as glycine, cysteine or serine; and pyruvate can be converted to alanine or enter into

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biosynthesis of fatty acids by its conversion into citrate in the mitochondria (Figure 5). In this

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regard, the absence or inhibition of the glucose-6-phosphate dehydrogenase enzyme (G6pd), a

key enzyme in the pentose phosphate pathway, impairs self-renewal and favors endodermal

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(Manganelli et al., 2012) and myogenic (Bracha et al., 2010) differentiation of mouse stem cells, respectively. To maintain pluripotency ESCs have to guarantee a proper flux of metabolites to

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ensure membranes, nucleic acids and protein biosynthesis, necessary for cell proliferation. Any

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impair ESC self-renewal.

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external or internal alteration that could alter the proper function of these metabolic pathways may

Under conditions of high cell proliferation, the Krebs cycle also supplies intermediaries to other anabolic pathways. The flow of metabolites from the Krebs cycle to other biosynthetic pathways is known as cataplerosis. For example, fatty acids are synthesized from acetyl-CoA that enters the mitochondria in the form of pyruvate and exists in the form of citrate (Figure 5). The biosynthesis of fatty acids is a fundamental biosynthetic route in ESCs. Fatty acids are necessary for the biosynthesis of cell membranes. ESCs (Moussaieff et al., 2015; Vazquez-Martin et al.,

2013) and NSCs (Knobloch et al., 2013) have a high expression of the genes encoding for the enzymes ATP-citrate lyase (Acly), Acetyl-CoA carboxylase (Acc) and fatty acid synthase (Fasn), three crucial enzymes in this biosynthetic pathway. The absence of Fasn in mouse NSCs impairs neurogenesis and proliferation of neural progenitors (Knobloch et al., 2013) and the chemical 24

Journal Pre-proof inhibition of Acc and Fasn impairs acquisition of pluripotency during cell reprogramming (VazquezMartin et al., 2013). Interestingly, the silencing or inhibition of Acly (Bauer et al., 2005; Hatzivassiliou et al., 2005), Acc (Brusselmans et al., 2005; Chajes et al., 2006) or Fasn (De Schrijver et al., 2003; Menendez et al., 2005) dramatically reduces the proliferation and survival of tumor cells in vitro. In addition, exported citrate from mitochondria to cytoplasm is essential to provide acetyl-CoA for protein acetylation (Wellen et al., 2009). Protein acetylation is a reversible post-translational

modification

catalyzed

by

lysine

acetyltransferases

(KAT)

and

lysine

deacetylases (KDAC), such as the Sirtuin family. The high rate of citrate-derived acetyl-CoA

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production in primed ESCs is key for maintaining the histone acetylation pattern (reviewed by Harvey et al., 2016a). The inhibition of glycolysis or Acly reduces cytosolic acetyl-CoA levels and

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histone acetylation, impairing pluripotency and favoring cell differentiation of human and mouse primed ESCs (Moussaieff et al., 2015) and mouse myoblasts (Bracha et al., 2010). In this regard,

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histone acetylation of H3K9 predicts pluripotency and reprogramming capacity of ES cells and

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histone deacetylase inhibitors, such as butyrate or valproic acid, support self-renewal of mouse and human ESCs, promotes their convergence toward a more naïve stage (Ware et al., 2014;

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Ware et al., 2009) and facilitates cell reprogramming (Huangfu et al., 2008). Acetyl-CoA is not only important for nuclear acetylation, but also for cytoplasmic post-translational modifications.

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Interestingly, high levels of acetyl-CoA promote acetylation and degradation of Fis1, decreasing

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mitochondrial fission. Conversely, a decrease in the levels of cellular acetyl-CoA or an increase in lipid generation, as a result of the Acc activation, lead to greater mitochondrial fission and cell reprogramming (Wang et al., 2017). These seemingly contradictory data suggest that a proper compartmentalization of metabolites in subcellular locations are essential for cell fate control (Figure 5). In fact, subcellular mitochondrial localization changes significantly throughout oocyte maturation and early embryo development (Van Blerkom, 2009). Given the role of metabolic intermediates derived from mitochondrial metabolism in regulating the epigenome, it has been

proposed that mitochondrial trafficking may help to maintain a proper metabolic communication with the nucleus (Harvey, 2019).

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Figure 5. Relevant metabolites and metabolic pathways for maintenance and acquisition of pluripotency. ESCs and iPSCs maintain a high glycolytic rate for non-exclusive energetic reasons: glycolysis is crucial to provide intermediaries of some anabolic pathways to favor the biosynthesis of nucleotides, membranes and proteins. The substrates marked in red correspond to those metabolites of glycolysis that act as intermediaries of other biosynthetic pathways. Besides, pyruvate-derived lactate provides NADH to feedback glycolysis with reduction power. Citrate depletion from the Krebs cycle for fatty acid biosynthesis jeopardizes the proper functioning of the Krebs cycle. The oxidation of glutamine maintains an adequate metabolic rate in the Krebs cycle, guaranteeing a proper αketoglutarate/succinate ratio, ATP production (OXPHOS) when necessary, and fatty acids synthesis, due to the fact that α-ketoglutarate, produced by glutaminolisis, can be transformed to both succinate and citrate through the inverse reactions of the Krebs cycle. NAPDH required for fatty acid biosynthesis is not only provided by glucose-6-phosphate dehydrogenase (G6pd), but also by glutamine-derived malate through malic enzyme (ME). Some of the key enzymes for metabolic change that these cells undergo are shown: Acc, acetyl-CoA carboxylase; Acly, ATP-citrate lyase; G6pd,; Gdh, glutamate dehydrogenase; Gls, glutaminase; Fasn, fatty acid synthase; Hat, Histone acetyltransferase; Hk,

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Journal Pre-proof hexokinase; Jhdm, Jumonji-C domain-containing histone demethylase; Ldh, lactate dehydrogenase; Mdh, malate dehydrogenase; Pdh, pyruvate dehydrogenase; Pfk, phosphofructokinase; Pk, pyruvate kinase; Tet, Ten-eleven translocation dioxygenase. ETC, electronic transport chain; α-KG, α-ketoglutarate.

Citrate depletion from the Krebs cycle for fatty acid biosynthesis jeopardizes the proper functioning of the cycle (Lunt and Vander Heiden, 2011). In addition, it has been described that human ESCs cannot use pyruvate-derived citrate efficiently due to a poor expression of isocitrate dehydrogenase 2/3 (IDH2/3) and aconitase 2 (ACO2) (Tohyama et al., 2016). Also, human primed ESCs have a high expression of uncoupling protein 2 (UCP2), which favors retrograde transport of

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pyruvate from the mitochondria to the cytoplasm, therefore reducing oxidative phosphorylation rates (Vozza et al., 2014). This protein tends to be silenced during human ESCs differentiation to favor metabolic conversion towards the oxidative state (Zhang et al., 2011). Glutaminolysis is an

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anaplerotic route that not only guarantees the functioning of the Krebs cycle in the absence of

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some Krebs cycle intermediates, but also provides a nitrogen source and NADPH for purine and

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pyrimidine biosynthesis in the cytosol (DeBerardinis et al., 2008) (Figure 5). The enzyme glutamine synthetase (GS) is essential during the early stages of embryogenesis (He et al., 2007)

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and the presence of glutamine in the cell culture medium increases preimplantation embryo viability by 46% (Rezk et al., 2004) (Figure 4). The oxidation of glutamine maintains an adequate

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metabolic rate in the Krebs cycle, guaranteeing a proper α-ketoglutarate/succinate ratio, the ATP

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production (OXPHOS) when necessary, and fatty acids synthesis, due to the fact that α-

ketoglutarate, produced by glutaminolisis, can be transformed to both succinate and citrate through the inverse reactions of the Krebs cycle by IDH and ACO enzymes (Metallo et al., 2011; Mullen et al., 2011). However, the degree of glutamine dependence varies according to the pluripotent state, highlighting that the cellular context and cellular maturity can alter the effect of glutamine. In this sense, bivalent (OXPHOS/glycolysis) naïve ESCs can oxidize both, glucose and glutamine (Figure 4), therefore they can be grown without glutamine for longer periods than glycolytic primed ESCs,

which cannot use pyruvate efficiently for maintaining a proper function of Krebs cycle (Tohyama et al., 2016; Vardhana et al., 2019). Besides, mouse naïve ESCs exhibit a higher proportion of αketoglutarate/succinate than primed ESCs (Vardhana et al., 2019). A reduction in mitochondrial citrate levels affects α-ketoglutarate/succinate ratios and fluctuations in these ratios can negatively 27

Journal Pre-proof affect epigenetic maintenance of ESCs, impairing self-renewal (reviewed by Harvey et al., 2016a). α-Ketoglutarate is a crucial substrate of the family of ten-eleven translocation (Tet) dioxygenases and the Jumonji-C domain-containing histone demethylase (Jhdm) family of proteins that catalyze DNA and histone demethylation, respectively (Su et al., 2016; Teperino et al., 2010) (Figure 5). A high α-ketoglutarate/succinate ratio promotes demethylation of histones and DNA, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression (Carey et al., 2015). In this regard, knockdown of several enzymes of Jhdm family impairs self-renewal of mouse ESCs and iPSCs

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(Das et al., 2014; Loh et al., 2007). Specifically, reduction of α-ketoglutarate levels in response to the knockdown of phosphoserine aminotransferase 1 decreases DNA 5′-hydroxymethylcytosine

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levels and increases histone methylation of naïve core transcriptional factor, such as Oct4, Sox2, Nanog, Klf4 and Esrrb, downregulating those genes, impairing mouse ESC self-renewal and

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inducing cell differentiation (Hwang et al., 2016). Conversely, high levels α-ketoglutarate or α-

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ketoglutarate/succinate ratio can promote early differentiation in human primed ESCs. Specifically, α-ketoglutarate induces and succinate impairs global histone and DNA demethylation (TeSlaa et

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al., 2016). In addition, mouse ESCs in the presence of glutamine silence the expression of histone deacetylase 1 and DNA methyltransferases 1 and 3a, which favors adequate histone acetylation

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and demethylation levels in essential promoters for the maintenance of pluripotency, such as that

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of Oct4 (Ryu et al., 2015). Furthermore, the absence of glutamine reduces intracellular glutathione levels in human ESCs, increasing the levels of oxidative stress in these cells. Under these conditions, Oct4 tends to oxidize and degrade, impairing self-renewal and favoring differentiation (Marsboom et al., 2016). The existence of a different regulation of biosynthetic pathways and glutaminolysis in naïve and primed pluripotent conditions remains unsolved. Future research will have to elucidate whether an intermingled metabolic state exist in PSCs rather than just the disjunctive between oxidative and glycolytic states.

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Journal Pre-proof Metabolic reprogramming: from OXPHOS to glycolysis During embryonic development or in vitro differentiation of ESCs, a metabolic change from glycolysis to OXPHOS occurs. As primed ESCs, primed iPSCs have been shown to display a glycolytic metabolism (Folmes et al., 2013; Varum et al., 2011). Activation of cell proliferation during the stochastic phase of reprogramming changes the metabolic requirements of cells compared to somatic cells, generally less proliferative (Lunt and Vander Heiden, 2011). In parallel

to mitochondria remodeling in cells undergoing cell reprogramming, a metabolic transition from a

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somatic-oxidative state to the pluripotent-glycolytic state occurs during the cell conversion (Folmes et al., 2011; Panopoulos et al., 2012) (Figure 6).

Epigenetic, transcriptomic (Buganim et al., 2012; Polo et al., 2012) and proteomic (Hansson

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et al., 2012) analyses have shown changes in the expression of proteins involved in the

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reconfiguration of glucose metabolism during cell reprogramming. These include the regulation of

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genes that participate in either of the different stages of glycolysis, pentose phosphate pathway (Prigione et al., 2011; Varum et al., 2011) or electronic transport chain complexes assembly

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(Folmes et al., 2011; Hansson et al., 2012). Thus, cell types that have a higher glycolytic rates and lower oxidative capacity are more prone to cell reprogramming (Panopoulos et al., 2012). In that

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sense, induction of glycolysis by chemical compounds (Zhu et al., 2010), hypoxic conditions

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(Yoshida et al., 2009), p53 inhibition (Krizhanovsky and Lowe, 2009), deacetylation of SIRT2-

acetylated key glycolytic enzymes (Cha et al., 2017) or addition of glycolytic intermediates (Folmes et al., 2012) increase cell reprogramming, whereas inhibition of glycolysis or activation of OXPHOS reduces it (Folmes et al., 2011; Son et al., 2013a; Zhu et al., 2010). As in the maintenance of pluripotency, HIFs are crucial in the metabolic change from OXPHOS to glycolysis during the first phase of cell reprogramming by activating the expression of Pdk and pyruvate kinase (Pk) (Mathieu et al., 2014; Prigione et al., 2014). Several reports have described a correlation between

a mature and tubular mitochondrial morphology and an oxidative metabolism, and between a fragmented and immature mitochondrial morphology and an aerobic glycolytic status (Mishra and Chan, 2016). In this sense, Mfn1/2 knockdown favors not only mitochondrial fragmentation, but also glycolytic conversion and Hif1 activation through Ras-Raf-Erk pathway in early-stage 29

Journal Pre-proof reprogramming, In addition, hypoxia decreases Mfn1/2 expression, facilitating pluripotency acquisition and maintenance (Son et al., 2015). However, OXPHOS seems to be necessary during the reprogramming process (Figure 6): heteroplastic mutations in the mtDNA generate a dysfunctional OXPHOS and impaired cell reprogramming of human primary fibroblasts (Yokota et al., 2015). Other studies have described that a transient OXPHOS activation is necessary for cell conversion (Kida et al., 2015; Prigione et

al., 2014) and cellular intermediaries that undergo cell reprogramming display both glycolysis and

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OXPHOS activation (Prieto et al., 2018). Kida and collaborators identified that these cellular intermediaries of reprogramming displayed a transient increase in OXPHOS. Specifically, the authors show that an early and transient upregulation of estrogen-related nuclear receptors α and

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γ (ERRα/γ) are required for inducing an OXPHOS burst that is essential for mouse iPSC

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generation. Early upregulation of ERRs marks cells that are destined to be reprogrammed. In

agreement with these results, it has been shown that Zic3 and Esrrb (Sone et al., 2017) and c-Myc

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(Prieto et al., 2018) enhance the reprogramming efficiency by inducing the expression of both glycolytic and oxidative enzymes during the first phase of the process, generating a hybrid

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hyperenergetic state by increasing the metabolic activity of both routes. Interestingly, Sone and

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cols. manage to generate naïve pluripotent stem cells directly from somatic cells through a delicate balance of glycolysis and OXPHOS orchestrated by Zic3 and Esrrb. In this regard, Esrrb-mediated

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OXPHOS activation, but not Zic3-mediated glycolysis, is critical for the conversion of primed PSCs into the naïve state. Together, these studies demonstrate that the inhibition of OXPHOS reduces cell conversion, contrary to the conclusions raised from previous reports. In this regard and similarly to what it has been shown in cancer cells (Swinnen et al., 2005), an early inhibition of OXPHOS may activate Ampk and reduce the efficiency of cell reprogramming (Vazquez-Martin et al., 2012b). Nevertheless, whether this transient OXPHOS activation plays an important role during cell reprogramming or is only a byproduct of OSKM overexpression remains to be explored. We hypothesized that, similarly to cell differentiation, epigenetic remodeling necessary for cell reprogramming requires high levels of energy in form of ATP, in addition to many other mitochondria-derived metabolites, such as, acetyl-CoA, α-ketoglutarate, NADH or SAM. The 30

Journal Pre-proof OXPHOS burst early in cell reprogramming may be critical to satisfy this demand. Once the cell reaches a stable iPSC state, OXPHOS is truly dispensable. ESCs and iPSCs have high MMP (Armstrong et al., 2010; Chung et al., 2007; Prieto et al., 2018). During cell reprogramming, cells with both high levels of MMP and OXPHOS are prone to reprogramming (Liu et al., 2013; Prieto et al., 2018). In addition, mouse ESCs and iPSCs express high levels of the ATP synthase inhibitory factor (Atpif1) (Vazquez-Martin et al., 2013) and its

induction during cell reprogramming is necessary for cell reconversion (Prieto et al., 2018).

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Interestingly, it has been described that the combination of both factors, a functional electronic transport chain and the inhibition of ATP synthase by Atpif1, promotes proliferation and survival of colon cancer cells by causing hyperpolarization of mitochondria and an increase in ROS levels: the

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proton gradient is not consumed due to ATPIF1-mediated inhibition of ATP synthase, which

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hyperpolarizes mitochondria and favors the appearance of ROS (Formentini et al., 2012; Sanchez-

Arago et al., 2013; Santamaria et al., 2006). Interestingly, the increase in ROS levels (Esteban et

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al., 2010; Ji et al., 2014) have been described during the first days of cell reprogramming. High local amounts of ROS can stimulate cell proliferation and survival through activation of the Pi3k/Akt

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pathways (Kwon et al., 2004; Lee et al., 2002), inhibition of protein tyrosine phosphatase receptors

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(Meng et al., 2002) or nuclear translocation of NF-κB transcription factors (Chandel et al., 2000). Hence, an increase in ROS levels may favor the efficiency of cell reprogramming by stimulating

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cell proliferation (Figure 6). In fact, it has been shown that an increase in ROS levels is necessary for cell reprogramming in MEFs (Zhou et al., 2016). In human cells, this increase in ROS favors the metabolic transition by activating HIF through nuclear respiratory factor 2 (NRF2) (Hawkins et al., 2016), similar to that of tumor development in colon (Santacatterina et al., 2016). This increase in ROS levels may favor the appearance of DNA damage; however, Nrf2 activates the antioxidant response in cancer cells (Santacatterina et al., 2016), which may act as a negative feedback to reduce ROS levels and resume cell growth normally. Besides, antioxidant treatments increase the efficiency of cell reprogramming (Esteban et al., 2010) (Figure 6).

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Figure 6. A metabolic transition from a somatic-oxidative state to the pluripotent-glycolytic state occurs

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during cell reprogramming. ESCs and iPSCs display a high division rate. Proliferating cells have a metabolism very different from non-proliferating cells. Activation of cell proliferation during the stochastic phase of reprogramming

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changes the metabolic requirements of cells compared to somatic cells. Aerobic glycolysis is crucial to provide

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intermediaries for biosynthetic pathways (anabolism). However, OXPHOS is also necessary during the reprogramming process. ERRα/γ, Zic3, Esrrb and c-Myc induce a hybrid hyperenergetic state by increasing the metabolic activity of both routes. ROS favors the metabolic transition by activating HIF through NRF2. HIFs are crucial in the metabolic change by

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activating the expression of Pdk and Pk. SIRT2 impairs cell reprogramming by acetylation of key glycolytic enzymes. High local amounts of ROS can stimulate cell proliferation. A positive feedback through MMP and ROS is proposed.

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Δψm, mitochondrial membrane potential.

The role of c-Myc

The Myc family of transcription factors is essential for early embryogenesis (Laurenti et al., 2009). Although ectopic expression of c-Myc is not necessary for cell reprogramming, its presence in the Yamanaka cocktail increases efficiency and speed of the process (Nakagawa et al., 2008; Wernig et al., 2008). Until now, it was not shown that cell reprogramming could take place in the absence of endogenous Myc genes. Exogenous or endogenous c-Myc expression seems to play an important role in the early stages of cell reprogramming (Knoepfler, 2008; Polo et al., 2012; Sridharan et al., 2009; Zviran et al., 2019). An interesting hypothesis for future research is that

32

Journal Pre-proof variations of endogenous Myc expression, forced by OSK or chemical compounds, may be a determining factor to reach the state of induced pluripotency. c-Myc is a proto-oncogene. It has been suggested that c-Myc favors reprogramming by activating the cell cycle (Mikkelsen et al., 2008; Polo et al., 2012; Sridharan et al., 2009). Since reprogramming is a process of epigenetic reconversion that increases successively cycle after cycle, the activation of proliferation increases the efficiency of the process, facilitating the erasure

of the somatic epigenetic patterns and establishing that of the pluripotent state (Hochedlinger and

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Plath, 2009). One of the main requirements and consequences of cell differentiation is to slow down or halt cell division rate. Thus, a fine control of the cell cycle is necessary for both maintenance of pluripotency and ensuing cell differentiation efficiently. The absence of Myc

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induces cell growth arrest (Heikkila et al., 1987; Wickstrom et al., 1988), differentiation in murine

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ESCs (Cartwright et al., 2005) or a dormant cellular state, similar to that observed in embryonic

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diapause of some mammals, under very controlled conditions (Scognamiglio et al., 2016). The reactivation of the cell cycle during somatic cell reprogramming, coupled to metabolic

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and mitochondrial reorganization, depends on the presence of c-Myc (Prieto et al., 2018). c-Myc induced phosphorylation and mitochondrial recruitment of Drp1 to drive fragmentation of these

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organelles during cell reprogramming from MEFs. This phosphorylation of Drp1 upon OSKM

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expression was mediated by Erk1/2 and Cdk1/cyclin B complex (Prieto et al., 2018). Mitochondrial

fission during the G2/M phase is a necessary process for the progression of the cell cycle (Lee et al., 2014; Qian et al., 2012; Taguchi et al., 2007). In this regard, c-Myc is a classic activator of cyclin B expression in tumor cells (Menssen and Hermeking, 2002; Seo et al., 2008; Yin et al., 2001) and binds to the Cdk1 promoter together with other transcriptional activators to favor its expression (Born et al., 1994; Liu et al., 1998; Perna et al., 2012). In the absence of serum, c-Myc cannot stimulate cell proliferation, only cell growth, protein synthesis and glycolytic metabolism

(Schuhmacher et al., 1999). Also, serum stimulation does not induce cell cycle activation in the absence of c-Myc (Schlosser et al., 2005). There is a close relationship between both effects. This relationship suggests that, during cell reprogramming, c-Myc may induce optimal conditions for inducing cell proliferation by factors present in the cell culture medium, including serum and Lif. 33

Journal Pre-proof Silencing of the dual specificity phosphatase family 6 (Dusp6) may be one of these prerequisites (Hirsch et al., 2015; Marshall et al., 2011; Polo et al., 2012; Soufi et al., 2012). In this regard, lack of Dusp6 may favor the early activation of Erk1/2, which could be further enhanced by serum and Lif stimulation (Burdon et al., 1999; Ohtsuka et al., 2015). Erk1/2 activation, together with the assembly of the Cdk1/cyclin B complex, induces phosphorylation of Drp1-S579. This phosphorylation triggers mitochondrial fission and thus facilitates Mesenchymal-to-Epithelial Transition (MET) during the stochastic phase of cell reprogramming.

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The role of c-Myc in the metabolic switch observed during cellular transformation in cancer is well understood (DeBerardinis et al., 2008). Previous reports have suggested that c-Myc plays a similar role during cell reprogramming (Folmes et al., 2013; Polo et al., 2012; Sridharan et al.,

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2009; Zviran et al., 2019). Hence, it has been described that the miR-290/371-Mbd2-Myc axis

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favors cell reprogramming by activating aerobic glycolysis through the induction of Pk and Ldh as it

does in cancer (Cao et al., 2015). In addition, c-Myc elicits expression of different subunits of the

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mitochondrial complexes of the electronic transport chain and Atpif1, all necessary for early activation of OXPHOS and MMP hyperpolarization (Prieto et al., 2018). In fact, c-Myc is critical for

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a proper OXPHOS activity and MMP levels in somatic cells (Morrish et al., 2008). Interestingly, the

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role of c-MYC in the Yamanaka cocktail for human cells can be replaced by the RNA binding protein LIN28 (Yu et al., 2007). This protein has been described as a key effector in glucose

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metabolism (Zhu et al., 2011), playing an important role in the metabolic transition from somatic cells to iPSCs and from the naïve to primed state in ESCs (Zhang et al., 2016) (Figure 6). Although it is dispensable for cell reprogramming, Lin28 favors cell reconversion when is combined with OSKM and endogenous absence of Lin28 considerably reduces reprogramming efficiency (Hanna et al., 2009). Since c-Myc and Lin28 seem to play a positive feedback in each other (Viswanathan and Daley, 2010), future research will shed light on the role of each protein, separately or in combination, along the process of cell reprogramming. It has also been described that c-Myc favors biosynthesis of fatty acids (Edmunds et al., 2014; Morrish et al., 2010) and nucleotides (Morrish et al., 2009), induces glutaminolysis dependency (Yuneva et al., 2007) and transcriptional program (Gao et al., 2009; Wise et al., 34

Journal Pre-proof 2008), and interplays with HIFs (Dang et al., 2008; Kim et al., 2007) in somatic and cancer cells, which are key metabolic features for self-renewal of PSCs (see above, Figure 5 ). However, the specific control of these pathways by c-Myc during cell reprogramming is not yet elucidated. The large amount of similarities between cell reprogramming and cell transformation (Prieto and Torres, 2017) suggests that c-Myc may promote acquisition of induced pluripotency by orchestrating the coordination of these biochemical routes in a similar fashion (Goetzman and

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Prochownik, 2018).

Conclusion

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Given the similarities with ESCs, iPSCs have become one of the most powerful tools in biomedicine and biotechnology. Although iPSCs can be obtained from any cell type of the adult,

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cell reprogramming is a very inefficient process. The different results shown by different

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laboratories upon the expression of reprogramming factors in somatic cells (from mouse or human origin) may reflect the different cellular responses that are induced when they are ectopically

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expressed. In this regard, the final effect of OSKM expression may depend on the physiological conditions of the targeted cell, such as cell organization, cell cycle phase or metabolic state.

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Interestingly, cells that undergo efficient reprogramming constitute a subpopulation of privileged

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cells with an ultrafast cell cycle (Guo et al., 2014). Investigating cell cytoarchitecture, gene expression or metabolic profile of this subpopulation of cells would help to understand the nature of the intrinsic advantages that they present compared to the normal cell population. This review has illustrated how mitochondrial dynamics and intracellular metabolic status influence cell fate, how these dramatic changes are interconnected, and how these cellular characteristics may contribute to epigenetic changes that eventually end up in the complete reactivation of the endogenous core pluripotency network (Folmes and Terzic, 2016; Ryall et al., 2015; Wu et al., 2016). Mitochondrial fission, Erk signaling, MET and Myc-dependent metabolic remodeling are central processes in tumorigenesis. Data presented in this review also underscore the close parallelism between the early stages of cell reprogramming and cell transformation. The correct 35

Journal Pre-proof activation of the endogenous core circuitry of pluripotency during the deterministic phase of cell reprogramming will direct the cell into the induced pluripotent state. In addition, these similarities between both processes reveal that any progress in the control of induced pluripotency will not only help to manage adequately this powerful tool for its use in biomedical applications, but also to better understand initial stages of cellular transformation in human malignancies.

Conflicts of interest

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The authors declare that there are no conflicts of interest.

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Acknowledgments

This work was supported by grant BFU2015-68366-R MINECO/FEDER (UE) to Josema

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Torres. Javier Prieto was supported by postdoctoral fellowships from Fundación Alfonso Martin

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Journal Pre-proof Comparison of naive and primed pluripotent states

Epiblast state

Implantation state

2i/Lif

Fgf2/Activin A

Methylation status

Hipomethylation

Variable

XX chromosomes

XaXa

XaXi

Developmental potential

Unbiased

Variable

Teratoma formation

Yes

Yes

Chimeras formation

Yes

No

Pluripotency factors

Oct4, Sox2, Nanog, Klf2, Klf4, Esrrb, Tbx3, Zfp42

Oct4, Sox2, Nanog

Myc expression

Low

High

Fgf/Erk pathway

Differentiation

Self-renewal

Mitochondrial network

Fragmented

Fragmented

Metabolism

OXPHOS/Aerobic glycolysis

Aerobic glycolysis

Gln dependency

Medium

High

MMP

High

Very high

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Culture conditions

2

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Embryonic equivalent

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Primed PSCs

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1

Naive PSCs

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Property

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Journal Pre-proof Highlights: Somatic cells can be reprogrammed to pluripotency by either ectopic expression of defined factors or exposure to chemical cocktails. During reprogramming, somatic cells undergo dramatic changes in a wide range of cellular processes, such as metabolism, mitochondrial morphology and function, cell signaling pathways or immortalization. Regulation of these processes during cell reprograming lead to the acquisition of a pluripotent state, which enables indefinite propagation by

symmetrical self-renewal without losing the ability of reprogrammed cells to differentiate into all cell types of the adult. In this review, recent data from different laboratories showing how these

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processes are controlled during the phenotypic transformation of a somatic cell into a pluripotent

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stem cell will be discussed.

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