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Research Article Changes in the Expression of Mitochondrial Morphology-Related Genes During the Differentiation of Murine Embryonic Stem Cells

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Research Article Changes in the Expression of Mitochondrial Morphology-Related Genes During the Differentiation of Murine Embryonic Stem Cells Hindawi Stem Cells International Volume 2020, Article ID 9369268, 12 pages https://doi.org/10.1155/2020/9369268 Research Article Changes in the Expression of Mitochondrial Morphology-Related Genes during the Differentiation of Murine Embryonic Stem Cells Jeong Eon Lee,1 Bong Jong Seo,1 Min Ji Han,1 Yean Ju Hong,1 Kwonho Hong,1 Hyuk Song,1 Jeong Woong Lee ,2 and Jeong Tae Do 1 1Department of Stem Cell and Regenerative Biotechnology, KU Institute of Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea 2Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea Correspondence should be addressed to Jeong Tae Do; [email protected] Jeong Eon Lee and Bong Jong Seo contributed equally to this work. Received 22 August 2019; Revised 17 December 2019; Accepted 7 January 2020; Published 28 January 2020 Academic Editor: Heinrich Sauer Copyright © 2020 Jeong Eon Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is mediated by proteins encoded by Fis1 and Dnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion- and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression of Mfn2 was increased and that of Dnm1L was decreased as expected, the other exceptions being Mfn1, Opa1, and Fis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission- related genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology according to the extent of differentiation. 1. Introduction [7]. For example, Folmes et al. showed that the globular shape of mitochondria progressively changed to elongated During embryonic development, cells undergo various during embryonic development from zygote to somite changes in gene expression [1, 2] and signaling pathways embryo. Accordingly, metabolic features such as pyruvate [3]. Metabolism and intracellular organelle structures are oxidation, glucose oxidation, glycolysis, and the pentose also altered during development and differentiation [4–6]. phosphate pathway (PPP) were also changed dynamically Specifically, the organellar changes are observed during the [6]. These features return to the developmental early-stage regaining of pluripotency (also known as reprogramming) status during the reprogramming process [8]. Some of the 2 Stem Cells International most dramatic changes in cells during development and dif- differentiate, they require more energy to meet the demands ferentiation occur in the mitochondria, which play essential of their newly acquired functions [6]. roles in cellular processes, including energy metabolism [9], Accordingly, mitochondria undergo dynamic remodeling apoptosis [10], aging [11], reactive oxygen species produc- during differentiation, and thus, mitochondrial morphology tion, calcium homeostasis, and differentiation [12]. and metabolism are changed. Therefore, we hypothesized Mitochondria continuously change their morphology that expression of mitochondrial fusion- and fission-related through fusion and fission in response to cellular require- genes may be changed toward a certain direction during the ments, which is the crux of mitochondrial quality control. spontaneous differentiation of ESCs. Here, we quantified In addition, mitochondria increase their population through the expression levels of the fusion-related genes Mfn1, self-division from the existing mitochondria; this is called Mfn2, and Opa1 and the fission-related genes Fis1 and mitochondrial biogenesis [13–16]. Mitochondrial dynamics Dnm1L during the differentiation of murine ESCs. Here, we and biogenesis vary by cell type and cellular environment. investigated these genes to determine whether they could be In addition, the quality and quantity of mitochondria can used as an index of the extent of differentiation and changes affect the cellular behavior and play a pivotal role in cell in mitochondrial morphology. metabolism [17]. In preimplantation embryonic and pluripotent stem 2. Materials and Methods cells, immature mitochondria characterized by a small and globular shape with poorly developed cristae are observed All methods used in this study were carried out in accordance [14, 18, 19]. Cells with immature mitochondria show low with animal care and use guidelines, and all experimental oxygen consumption and high levels of glycolytic enzymes protocols were approved by the Institutional Animal Care [20]. Thus, undifferentiated embryonic stem cells (ESCs) also and Use Committee of Konkuk University. exhibit low levels of ATP production, modest levels of antiox- idant enzymes, and poor oxidant capacity [14, 19, 20]. Upon 2.1. Cell Cultures. Mouse ESCs (E14tg2a) were purchased differentiation of ESCs, mitochondria in these cells become from the American Type Culture Collection (ATCC, Manas- elongated, showing developed cristae and dense matrices sas, VA, USA) and cultured on culture dishes layered with [21]; this results in high oxygen consumption and ATP pro- inactivated MEFs in an ESC medium, consisting of Dulbec- duction for more efficient cellular activity [14, 18, 19]. co’s modified Eagle’s medium (DMEM; Gibco) supple- In mammals, mitochondrial morphology switches mented with 15% fetal bovine serum (FBS; HyClone), 1× between elongated tubular and fragmented globular by Penicillin/Streptomycin/Glutamine (P/S/G; Gibco), 0.1 mM fusion and fission, respectively [22, 23]. Mitochondrial fusion nonessential amino acids (NEAAs; Gibco), and 1 mM β-mer- is mediated by the dynamin family GTPases, such as mitofu- captoethanol (Gibco) with 1000 U/mL leukemia inhibitory sin (MFN) 1, MFN2, and optic atrophy 1 (OPA1) [24–26]. factor (ESGRO, Chemicon International). MEFs were cul- Although the exact fusion mechanism has yet to be defined, tured in culture dishes coated with 0.15% porcine gelatin MFN1 and MFN2 form a dimer that inserts itself into the (Sigma, St. Louis, MO, USA) in MEF medium consisting of mitochondrial outer membrane, whereas OPA1 is located DMEM (Gibco) supplemented with 15% FBS (HyClone), in the mitochondrial inner membrane [27, 28]. MFN1, 1x P/S/G (Gibco), 0.1 mM NEAAs (Gibco), and 1 mM MFN2, and OPA1 contain a GTPase domain, hydrophobic β-mercaptoethanol (Gibco). NSCs were cultured in culture heptad repeat (HR) domain, and transmembrane domain dishes coated with 0.15% porcine gelatin (Sigma) in NS [24, 29]. MFN1 and MFN2 play similar roles in mitochon- medium consisting of DMEM : Nutrient Mixture F-12 drial fusion and thus can functionally replace each other (Gibco), 0.5 mg/mL bovine serum albumin (BSA; Sigma), and form homotypic or heterotypic dimers [25, 30]. 1% N2 supplement (Gibco), 1x NEAAs (Gibco), 1x P/S/G In contrast, the major proteins related to mitochondrial (Gibco), 10 ng/mL basic fibroblast growth factor (bFGF; fission are FIS1 [31–33] and dynamin-related protein 1 R&D systems), and 10 ng/mL epidermal growth factor ° (DNM1L, also called DRP1) [34–36]. DNM1L is mainly (EGF; Gibco). All cell lines were incubated at 37 Cinan located in the cytosol and recruited to the outer membrane atmosphere of 5% CO2 and maintained on tissue culture of the mitochondria where it induces fission [30]. FIS1 is dishes (Corning, Amsterdam, The Netherlands). located in the outer mitochondrial membrane and is closely related to DNM1L [32, 33]. DNM1L can interact with other 2.2. In Vitro Differentiation. Pluripotent ESCs maintain mitochondrial fission proteins, including mitochondrial fis- stemness in the presence of LIF via the JAK-STAT3 pathway. sion factor (MFF) and FIS1. Interestingly, a recent study However, when this pathway is inhibited in response to LIF suggested that DNM1L can interact with the fusion protein withdrawal, pluripotent ESCs randomly differentiate into MFN and facilitate MFN-mediated fusion [30]. Although endodermal, ectodermal,
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