ReviewMitochondrial dynamics and metabolism in induced pluripotency
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 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 development of induced pluripotent stem cells (iPSCs) one of the most important advances in the field of biology in the last two decades (Takahashi and Yamanaka, 2016).
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 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 transfer (Cibelli et al., 1998; Munsie et al., 2000; Wakayama et al., 2001). Among the different 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 (Fig. 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 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 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 used for cell reprogramming (Fig. 1). 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 (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 numerous cells are refractory to cell reprogramming. These refractory cells are trapped in cellular intermediaries along the process (Fig. 1).
Self-renewal is the ability of stem cells to give rise to exact copies of themselves. Both ESCs 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.
Section snippets
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 (Fig. 2). When fission is greater than fusion, equilibrium shifts
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 strategy display different features: timing, epigenetic remodeling and somatic-to-pluripotency developmental routes (Cacchiarelli et al., 2015; Takahashi et al., 2014; Velychko et al., 2019; Zhao et al., 2015). However, cell reprogramming and cell differentiation always have opposite developmental-start and -end points. In
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 development, ranging from zygote to gastrulation stage, a profound reorganization of cellular metabolism and mitochondrial dynamics takes place.
During the states of oocyte and fertilized egg, energy is primarily obtained by oxidation of pyruvate and lactate, rather than glucose (Brinster and Troike, 1979; Downs
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 (Fig. 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
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
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
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
Conclusion
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, cell reprogramming is a very inefficient process. The different results shown by different 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 expressed. In this regard, the
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by grant BFU2015-68366-R MINECO/FEDER (UE) to Josema Torres. Javier Prieto was supported by postdoctoral fellowships from Fundación Alfonso Martin Escudero and Generalitat Valenciana (VALi+d 2019 program).
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