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  • Introduction The majority of induced pluripotent

    2018-11-08

    Introduction The majority of induced pluripotent stem cells (iPSCs) are not fully reprogrammed (Chan et al., 2009; Lee et al., 2013; Mikkelsen et al., 2008). There is still minimal information on transition checkpoints necessary to induce full reprogramming. Pluripotent stem cells (PSCs) have functions and requirements different from somatic cells. In contrast to somatic cells, PSCs have a shorter G1 phase of the cell cycle and unlimited cell growth capacity, as in cancer stem cells (CSCs). While PSCs have metabolic activities very different from those of somatic cells to meet a proper balance between ATP production as an energy source and biosynthetic demands (Varum et al., 2011; Zhang et al., 2012), metabolic differences between partial (par) and fully (full) reprogrammed iPSCs are yet to be determined. Increased glycolytic flux is characteristic of CSCs and embryonic stem cells (ESCs). They require increased ATP and anabolic precursors for rapid proliferation compared with normal somatic cells (Zhang et al., 2012). Metabolism of ESCs differs from CSCs; they contain lower numbers of mitochondria and less mtDNA (Folmes et al., 2012), and have less active oxidative phosphorylation (Folmes et al., 2011). ESCs are particularly vulnerable to oxygen radicals, which cause genomic damage, apoptosis, and decreased differentiation capacity (Han et al., 2008; Zhang et al., 2012). This is important for lower mitochondrial respiration in PSCs, because reactive oxygen species (ROS) generation is a consequence of electron transport chain (ETC) activity. Shifting to cysteine protease inhibitors glycolysis is a form of ROS defense. Somatic cells must switch mitochondrial oxidative metabolism to anaerobic glycolytic pathways during iPSC reprogramming (Folmes et al., 2011; Liu et al., 2013; Xu et al., 2013). However, little is known regarding molecular mechanisms involved in metabolic reprogramming. We reported that CD34+ cells from thawed human cord blood (CB) cryopreserved for more than 20 years could be successfully reprogrammed using lentiviruses expressing Yamanaka factors (Broxmeyer et al., 2011). Here, we identified partially reprogrammed cells by cell-surface expression of TRA-1-60, a bona fide marker of fully reprogrammed cells (Chan et al., 2009). Twelve days after CD34+ cells were transduced with Yamanaka factors, about 5% of total colonies expressed TRA1-60. These colonies represented fully reprogrammed cells (Lee et al., 2013). We also observed TRA1-60-negative colonies, with morphology similar to that of human ESCs (hESCs) but representing partially reprogrammed cells. We suggested that partially reprogrammed cells are “trapped” in an incompletely reprogrammed yet stable state not sufficient for full reprogramming (Chan et al., 2009; Lee et al., 2013). We hypothesized that shifts in mitochondrial metabolic pathway checkpoints might be central to early stages of reprogramming prior to full pluripotency induction. Evidence for this exists (Folmes et al., 2011; Panopoulos et al., 2012; Xu et al., 2013), but detailed molecular mechanisms are lacking. MicroRNAs (miRNAs) regulate biological processes including embryonic development and somatic cell reprogramming (Anokye-Danso et al., 2011; Bang and Carpenter, 2008; Bartel, 2004). Among miRNAs upregulated in fully reprogrammed iPSCs (Lee et al., 2013), we focused on miRNA 31 (miR-31), which is involved in cell proliferation (Slaby et al., 2007), migration (Valastyan et al., 2009), apoptosis (Bhatnagar et al., 2010), and differentiation (Deng et al., 2013). We postulated a role for miR-31 in metabolic transition to fully programmed iPSCs (fulliPSCs). Using bioinformatics, luciferase reporter assays, and overexpression experiments, a highly conserved binding site for miR-31 was predicted in the 3′ UTR of succinate dehydrogenase (succinate dehydrogenase complex subunit A; SDHA), which is part of the mitochondrial respiratory chain complex II and is encoded by nuclear DNA (Grimm, 2013), and participates in electron transfer in the respiratory chain and succinate catabolism in the Krebs cycle (Kim et al., 2012). Mutations in SDHA are rare but are linked to severe metabolic disorders resulting from decreased Krebs cycle activity, impaired oxidative phosphorylation, and bioenergetic deficiency. In contrast to other SDHs, inhibition of SDHA does not increase production of ROS under conditions of normal O2 tension (normoxia), suggesting that reduction of SDHA activity might be an ideal way to reduce mitochondrial ETC activity, thus promoting fulliPSC induction while maintaining low ROS generation (Guzy et al., 2008). Evaluating metabolism of iPSCs without dissecting fully from partially reprogrammed iPSCs (pariPSCs), where the partially reprogrammed cells represent a majority of iPSCs, does not allow accurate evaluation of regulation of fulliPSC metabolism. We identified an miR-31/SDHA axis involvement in conversion of somatic cells to fulliPSCs, delineating a previously unrecognized role for miR-31 in regulating mitochondrial metabolism through suppressing SDHA during reprogramming. This may serve as a stratagem to more efficiently generate fulliPSCs for potential therapeutic use.