Changes in mitochondrial thymidine metabolism and mtDNA copy number during induced pluripotency

Changes in mitochondrial thymidine metabolism and mtDNA copy number during induced pluripotency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Changes in mitochondrial thymidine metabolism and mtDNA copy number during induced pluripotency Man Ryul Lee, Hyun Kyu Kim, Yena Song, Minji Kye, Byeongho Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5148938/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jun, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 12 You are reading this latest preprint version Abstract Reprogramming somatic cells into human induced pluripotent stem cells entails profound intracellular changes, including modifications in mitochondrial metabolism and a decrease in the mitochondrial DNA copy number. However, the mechanisms underlying this decline in mitochondrial DNA copy number during reprogramming remain unclear. In this study, we aimed to elucidate these underlying mechanisms. Through meta-analysis of numerous RNA sequencing datasets, we identified the genes responsible for the reduction in mitochondrial DNA. We investigated the functions of these identified genes and examined their regulatory mechanisms. Particularly, the thymidine kinase 2 ( TK2 ) gene, required for mitochondrial DNA synthesis and found in the mitochondria, exhibits diminished expression in human pluripotent stem cells compared with that in somatic cells. TK2 was substantially downregulated during reprogramming and markedly upregulated during differentiation. Collectively, the reduction in TK2 levels influences a decrease in mitochondrial DNA copy number and participates in shaping the metabolic characteristics of human pluripotent stem cells. However, contrary to our expectations, treatment with a TK2 inhibitor impaired somatic cell reprogramming. These results suggest that reduced TK2 expression may result from metabolic conversion during somatic cell reprogramming. Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Stem cells/Reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Induced pluripotent stem cells (iPSCs) are reprogrammed cells obtained by introducing reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) into somatic cells, leading them through stochastic and determinate phases to acquire pluripotency 1 , 2 , 3 , 4 . Reprogramming orchestrates dynamic shifts in gene expression profiles, epigenetic landscapes, metabolic traits, and cellular morphology within somatic cells 5 , 6 . Despite comprehensive investigations, the acquisition of pluripotency from somatic cells during the stochastic phase is acknowledged as a sluggish and inefficient process owing to its intricate nature 2 . Endeavors to illuminate the intricacies of reprogramming persist; however, the underlying metabolic mechanisms pivotal for acquiring pluripotency remain largely unknown. To ensure an essential energy supply for cell survival, cells typically break down glucose into pyruvate in the presence of oxygen, subsequently subjecting it to oxidative phosphorylation (OXPHOS) within the mitochondria to synthesize ATP. However, even in the presence of sufficient oxygen, pluripotent stem cells (PSCs) engage in aerobic glycolysis to convert pyruvate into lactate instead of transporting pyruvate to the mitochondria for ATP production. This process is relatively inefficient from an energy production perspective. However, in cases where the metabolic flux is sufficiently high, and during rapid cellular proliferation that characterizes undifferentiated embryonic stem cells (ESCs) and iPSCs, aerobic glycolysis can be considered a favorable metabolic mechanism 7 . Aerobic glycolysis can produce a rapid supply of ATP and is recognized as a pivotal process that provides essential building blocks, including nucleic acids generated through the pentose phosphate pathway, amino acids, and lipids required for biosynthesis, particularly during the observed robust proliferation at the initial stages. Therefore, the process by which somatic cells acquire pluripotency through reprogramming involves an essential concomitant transition from the predominantly occurring somatic cell metabolism, characterized by OXPHOS, to the metabolic profile characteristic of PSCs, namely, aerobic glycolysis 8 , 9 , 10 , 11 . During reprogramming, it has been reported that the fundamental mechanism behind metabolic changes involves the upregulation of glycolytic genes, contributing to the activation of this process, while concurrently downregulating mitochondrial respiratory chain complex genes to enhance reprogramming efficiency 8 , 9 , 10 , 11 , 12 , 13 , 14 . Additionally, morphological alterations in the mitochondria influence reprogramming efficiency 10 , 11 , 15 , 16 , 17 . During reprogramming, mature tubular and cristae-rich mitochondria found in somatic cells transition into immature spherical mitochondria with reduced cristae as pluripotency is acquired 16 , 18 , 19 . These morphological and functional changes in mitochondria are closely associated with the acquisition of metabolic traits in PSCs and directly affect reprogramming efficiency 15 , 16 , 17 , 18 , 20 . Notably, PSCs, characterized by aerobic glycolysis, exhibit a marked decrease in mitochondrial DNA (mtDNA) copy numbers compared with that in somatic cells 10 , 21 , 22 . mtDNA, a circular DNA molecule spanning 16.6 kb, encodes 13 essential protein subunits of mitochondrial respiratory chain complexes I, III, IV, and V, as well as 22 tRNAs and 2 rRNAs required for the translation of mitochondrial subunits 23 , 24 . Therefore, changes in the mtDNA copy number can directly affect mitochondrial function and the acquisition of pluripotency during reprogramming. The regulation of mtDNA copy number is tightly controlled by nuclear-encoded mtDNA replication factors 25 . Loss of function of genes involved in mtDNA replication and synthesis leads to decreased mtDNA copy number, mitochondrial defects, and increased reliance on aerobic glycolysis, resembling the Warburg effect 26 . Similarly, metabolic conversion from OXPHOS to glycolysis also occurs during somatic cell reprogramming and is accompanied by a decrease in mtDNA copy number 10 , 22 . Furthermore, human ESCs have a relatively low number of mtDNA copies compared with somatic cells 16 . Contrastingly, mitochondria undergo marked changes during cellular differentiation, leading to an increase in mtDNA copy number and a mature structural state characterized by a dense matrix, complex cristae, and dispersed localization in the cytoplasm 16 , 17 , 18 , 19 . This suggests that mtDNA copy number is closely linked to the identity of stem cells during induced pluripotency. However, the molecular mechanisms underlying alterations in mitochondrial function and mtDNA copy number are not well understood. In this study, we focus on the differential expression of thymidine kinase 2 (TK2) in somatic cells and pluripotent stem cells (PSCs), and its potential role in regulating mitochondrial DNA (mtDNA) copy number and the acquisition of pluripotency characteristics. Human cells possess two enzymes, TK1 and TK2, that are involved in phosphorylating thymidine, a building block of DNA necessary for DNA replication and synthesis. TK1 demonstrates cell cycle-dependent activity in the cytoplasm, showing high activity in rapidly dividing cells, including stem cells and tumor cells, whereas it is ubiquitinated and degraded in slow-dividing cells or cells with less active DNA replication 23 . In contrast, TK2, encoded by a gene located on chromosome 16, is translated and targeted to the mitochondria, where it functions independently of the cell cycle and is involved in mitochondrial thymidine synthesis 10 , 20 , 21 . This distinction in the roles of TK1 and TK2 suggests a unique metabolic regulation in PSCs and their mitochondrial function. This study aims to explore the role of TK2 in mtDNA synthesis and its contribution to pluripotency. We intend to investigate the expression levels of TK2 during the reprogramming process and identify potential genetic regulators of TK2 that influence mtDNA copy number. These insights will help further our understanding of the metabolic characteristics that are essential for maintaining stemness and enhancing reprogramming efficiency in PSCs. Material and Methods Culture and maintenance of human ESCs (hESCs) The hESC cell lines, H1 and H9 (WiCell Research Institute, Madison, WI, USA), and the hiPSC cell lines CMC-hiPSC-003, 009, and 011 (Korea Centers for Disease Control and Prevention, Osong, Korea), were maintained in complete TeSR-E8 medium (StemCell Technologies, Vancouver, Canada) for feeder-free culture. PSCs were plated on vitronectin-coated dishes. The medium was replaced every 24 h. For passaging, the cells were enzymatically detached using TrypLE Express (Gibco, Carlsbad, CA, USA) and transferred to a new coated dish every 5 d. Passages were prepared at split ratios of 1:5 or 1:10 The fibroblast cell lines BJ1, MRC5, and NT2 (human embryonal carcinoma stem cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM high glucose; Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (Corning) and 100× penicillin-streptomycin (Corning). Fibroblasts and NT2 cells were routinely passaged every 4–5 d using TrypLE Express. All cells were maintained in an incubator at 37°C with 5% CO 2 . Embryoid body ( EB) generation to induce spontaneous in vitro differentiation For EB generation, clumps of undifferentiated hESCs were mechanically detached using glass pipettes. Subsequently, these clumps were seeded onto non-adhesive bacterial dishes in differentiation medium devoid of basic fibroblast growth factors. The cells were then allowed to spontaneously aggregate to form EBs. The differentiation medium was refreshed on a daily basis, comprised of DMEM/F12 (Invitrogen, Gibco) supplemented with 20% KnockOut Serum Replacement (Invitrogen, Gibco), 1 mM glutamine, 1% nonessential amino acids, 0.1% penicillin/streptomycin, and 0.1 mM beta-mercaptoethanol. Maintenance and reprogramming using secondary reprogramming system hIF-T fibroblasts for somatic cell reprogramming (from Davide Cacchiarelli; Broad Institute, Cambridge, MA, USA) were cultured in an optimized DMEM/F12 culture medium supplemented with 10% fetal bovine serum 27 . The reprogramming was performed using the TeSR™-E7™ Medium for Reprogramming (2-Component) containing Vitronectin XF and doxycycline hyclate (D9891; Sigma–Aldrich, St Louis, MO, USA), according to the manufacturer’s instructions. Primosin (InvivoGen; ant-pm-1) at 50 μg/mL was used to prevent microbial contamination during somatic cell reprogramming. hIF-T-derived iPSCs were cultured in TeSR™-E8™ medium (STEMCELL Technologies; 05990) or StemMACS™ iPS-Brew XF (Miltenyi Biotec; 130-104-368) containing Vitronectin XF. Optical microscopy The morphologies of the ESCs, iPSCs colonies, and fibroblasts were observed using an optical microscope (CKX53; Olympus, Tokyo, Japan). Cell images were captured using an eXcope X9 (Dixi Science, Daejeon, Korea). Electron microscopy Cells were cultured on a 100-mm culture dish, washed twice with PBS, and fixed with 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. The samples were lifted using a cell lifter (Corning), centrifuged (4,000 rpm, 4°C, 30 min) and stored at 4°C until further processing. The samples were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Eponate-12 resin (Ted Pella, Redding, CA, USA). One-micrometer-thick sections were prepared using a Reichert-Jung UltraCut E ultramicrotome (Reichert Technologies, Depew, NY, USA), stained with toluidine blue, and imaged (Olympus BX-51). Seventy-nanometer-thick sections per block were placed on formvar-coated slot grids, stained with uranyl acetate/lead citrate, and imaged using transmission electron microscopy (H-7600; Hitachi, Tokyo, Japan). Mito Stress Test The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer’s protocols. Fibroblasts were plated in wells of an XF96 cell culture microplate and incubated at 37°C in a CO 2 incubator for 24 h to ensure attachment. PSCs were plated in wells of an XF96 cell culture microplate coated with vitronectin XF (STEMCELL Technologies; 07180) and incubated at 37°C in a CO 2 incubator for 24 h to ensure attachment. Ten micrometers of Y-27632 was added to the culture medium only for the first 24 h after seeding. The assay was initiated after the cells were equilibrated for 1 h in XF assay medium supplemented with 10 mM glucose, 5 mM sodium pyruvate, and 2 mM glutamine in a non-CO 2 incubator. The substrate-based metabolic assay was performed by applying 10 mM glucose injections after starvation in XF DMEM (pH 7.4; Seahorse Bioscience). Mito Stress Test of the cells was employed before and after the sequential injection of 2 μM oligomycin, 1μM Carbonyl cyanide m‐chlorophenylhydrazone (CCCP), 0.5 μM rotenone, and 0.5 μM antimycin A. Each OCR and ECAR value was normalized after protein quantification using BCA Protein Assay Kit (Thermo Fisher Scientific, Wilmington, DE, USA; 23227) at the final step of the assay. RNA extraction, reverse transcription, and quantitative real-time PCR Cells were lysed with easy-BLUE (iNtRON, Daejeon, Korea) and total RNA was extracted. RNA was quantified using a NanoDrop spectrophotometer. One microgram of RNA was reverse-transcribed using the All-in-One 5× First Strand cDNA Synthesis Master Mix kit (CellScript, Madison, WI, USA), and quantitative PCR was performed using TOPreal™ qPCR 2X PreMIX (Enzynomics, Daejeon, Korea). The experiments were performed according to the manufacturer’s instructions. RNA was extracted from each treatment condition, and the experiments were performed in triplicate. Primer sequences were designed according to the Integrated DNA Technologies website (IDT; https://sg.idtdna.com/pages). All primers were validated for efficiency, and data were obtained from experiments performed in triplicate. Western blotting Cell lysates were extracted using NP40 (Elpis Biotech, Daejeon, Korea) and a 100× protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA;5872S). Cell lysates were centrifuged at 13,000× g for 20 min at 4°C, and the supernatants were obtained. The total protein concentration in each supernatant was measured using a BCA Protein Assay (Thermo Fisher Scientific; 23227). The protein samples were separated by electrophoresis using 8–12% SDS-polyacrylamide gels and transferred to 0.2-μM polyvinylidene fluoride blotting membranes (Amersham, Little Chalfont, UK). The membranes were blocked in 5% bovine serum albumin (BSA; BioShop, Burlington, Canada; ALB001.100) in TBS-T (50 mM Tris, 0.15 M sodium chloride, 0.05% Tween 20) for 1 h at 25°C. After blocking, the membranes were incubated with the primary antibody (1:1000) diluted in 1% BSA in TBS-T for 16 h at 4°C. The membranes were washed three times for 10 min with TBS-T at room temperature and then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2500) diluted in 1% BSA in TBS-T for 1 h at room temperature. Chemiluminescence was detected using a Pico EPD Western Blot Detection Kit (Elpis Biotech, EBP-1073) or Amersham ECL Prime Western Blotting Detection Reagent (Amersham, RPN2232). The antibodies used in this study are listed in Supplementary Table 1. Meta-analysis of gene expression data RNA-Seq data related to somatic cells, PSCs, and reprogramming were collected and downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, STAR aligner software was used to map the data onto the GRCh38 reference genome. Following mapping, the obtained read counts were used to analyze the expression levels of the genes of interest. The data (access codes) used for the analysis are listed in Supplementary Table 2. The results were visualized using R version 4.1.1. The ggplot2 R package was used to create violin plots for visualization. Reverse transcription quantitative PCR (RT-qPCR) Cells were lysed with easy-BLUE (iNtRON), and total RNA was extracted. The RNA was quantified with a Nanodrop ND-2000 spectrometer (Thermo Fisher Scientific). One microgram of the RNA was reverse-transcribed using the cDNA Master Mix (CellSafe, Yongin, Korea; CDS-100), and quantitative PCR was performed using TOPreal™ qPCR 2X PreMIX (Enzynomics;RT500M). cDNA synthesis and qPCR were performed according to manufacturer's instructions. Primer sequences were designed using the PCR primer design tool IDT (https://sg.idtdna.com/pages). All primers were validated for efficiency and specificity, and experiments were performed in triplicates. The primer sequences are listed in Supplementary Table 3. Mitochondrial DNA copy number analysis The cell pellet was lysed with 500 μL lysis buffer (Tris-HCL, 2 M pH 9.0; EDTA, 0.1 M pH 8.0; 0.5% SDS; and distilled water). Then, 10 μL 20 mg/mL Proteinase K Solution (Invitrogen; 25530049) was added, followed by an incubation for 3 h at 55°C or until the cell pellet was completely dissolved. Subsequently, following a treatment with RNase A (Thermo Fisher Scientific; EN0531, 10 mg/mL) at 65°C for 1 h, total cellular DNA was isolated using a conventional phenol-chloroform DNA extraction method. The DNA was dissolved in Tris-EDTA (TE) buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0). The DNA from all samples was diluted to 50 ng. To quantify mtDNA copy number, the nuclear DNA/mtDNA ratio was determined by employing the ND1 gene primer specific to mtDNA and the genomic DNA β-globin gene primer. The primer sequences are listed in Supplementary Table 4. Quantitative PCR was performed using TOPreal™ qPCR 2X PreMIX (RT500M; Enzynomics). Chromatin immunoprecipitation (ChIP) and qPCR ChIP analysis was conducted using a TruChIP Chromatin Shearing Kit (Covaris, Woburn, MA, USA; 520127). Initially, cells were subjected to a 3-min treatment with methanol-free 16% paraformaldehyde (Thermo Fisher Scientific; 28908) to enable fixation, followed by an isolation of the nuclei. Subsequently, the isolated nuclei were fragmented to approximately 500 bp using an next generation sequencing sample processor, M220 (Covaris). Immunoprecipitation was performed using the specific antibody and isotyped IgG, and the chromatin bound to the antibody was subsequently separated utilizing Dynabeads™ Protein G (Invitrogen). Precipitated chromatin was treated with proteinase K (Invitrogen; 25530049, 20 mg/mL) and reverse cross-linked by heating at 65°C for 4–5 h (or overnight). Then, after treatment with RNase A (Thermo Fisher Scientific; EN0531, 10 mg/mL) at 65°C for 1 h, DNA was purified using a conventional phenol-chloroform DNA extraction method. Finally, the target enrichment was assessed by quantitative analysis using qPCR. Information regarding the primer sequences used is provided in Supplementary Table 5. Analysis of somatic cell reprogramming efficiency Reprogramming samples were dissociated into individual cells using TrypLE™ Express Enzyme (1X) without phenol red (Gibco™). After harvesting and PBS washing, single-cell suspensions were permeabilized and fixed in a 4% paraformaldehyde solution (in PBS). The cells were labeled with SSEA4 (APC) and TRA-1-60 (FITC) antibodies, followed by washing. The fluorescence-activated cell sorting (FACS) antibodies used in this study are listed in Supplementary Table 1. All steps involving cell permeabilization, fixation, staining, and data acquisition were performed on the same day using the same instrument for each experiment to maintain consistency. Analysis and cell sorting were performed using a FACS Canto II instrument from BD Biosciences (Franklin Lakes, NJ, USA), and the data were processed using BD FACSCanto™ Clinical Software. Statistical analysis All experiments were performed three times in triplicate, and data are presented as mean ± standard deviation (SD) for statistical comparison. Statistical significance of differences between groups was evaluated using Student’s t-test. P values less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, Inc., San Diego, CA, USA). Results Down-regulation of TK2 gene expression and mtDNA copy number is a molecular signature of human pluripotency To examine the metabolic changes occurring during somatic cell reprogramming, we compared the morphological features of mitochondria between fibroblasts (MRC5) and PSCs (hiPSCs; CMC-003 and hESC; H1) using electron microscopy. The analysis revealed that fibroblasts displayed mitochondria with dense cristae and an elongated tubular shape, whereas PSCs exhibited mitochondria with fewer cristae and a round shape (Fig. 1a). To determine whether these morphological differences between fibroblasts and PSCs corresponded to functional disparities, we measured mitochondrial function and the OCR/ECAR ratio using Seahorse XF96 with a series of drug injections consisting of an ATP synthase inhibitor (oligomycin), an uncoupler (CCCP), and electron transport chain inhibitors (antimycin A and rotenone). Human PSCs displayed considerably lower overall OCR levels and OCR/ECAR ratios than fibroblasts (Fig. 1b and c). Specifically, during basal respiration or maximal respiration, the OCR in hPSCs was approximately 2–5 times lower than that in fibroblasts (Fig. 1b). In hPSCs, OCR linked to mitochondrial inner membrane proton leakage was approximately 11-fold lower than that in fibroblasts, and OCR linked to ATP production was also considerably lower (Fig. 1b). This result indicates that mitochondrial function and ATP production efficiency of hPSCs are relatively low compared to those of fibroblasts. Additionally, hPSCs exhibited a markedly reduced spare-respiratory capacity (calculated as maximum OCR minus basal OCR) compared to fibroblasts (Fig. 1b), indicating that hPSCs maintain morphologically immature mitochondrial features as well as functionally immature characteristics. One particularly noteworthy observation was the marked difference in mtDNA copy numbers between fibroblasts and PSCs (Fig. 1d). mtDNA encodes 13 genes that are critical for OXPHOS. Maintaining a certain mtDNA copy number is essential for mitochondrial function. Therefore, it can be hypothesized that the metabolic shift during reprogramming may be attributed to changes in mtDNA copy numbers, resulting in decreased mitochondrial function and acquired pluripotency. Identification of TK2 as a key regulator in mtDNA maintenance during reprogramming To identify specific genes related to mtDNA replication and synthesis, we conducted a meta-analysis using open RNA-seq databases (Fig. 2a). RNA-seq data for somatic cells, iPSCs, ESCs, and reprogramming processes were downloaded from the GEO database (Supplementary Table 2). After data mapping and normalization using a standardized pipeline, we analyzed the expression patterns of the target genes. The constructed database accurately reflected the expression of pluripotency markers, such as OCT4, SOX2, and NANOG (Fig. 2a). Most genes involved in mtDNA synthesis and replication were considerably downregulated during reprogramming, with TK2 showing the most notable decrease (Fig. 2b, Supplementary Table 1). We propose that for mtDNA synthesis to occur efficiently and emerge as a pivotal factor in mitochondrial metabolic transition, thymidine supply, primarily governed by TK2, takes precedence over other factors, thereby underscoring the importance of TK2 in the regulation of mtDNA copy numbers. Therefore, we aimed to elucidate the functional characteristics of TK2 during reprogramming. To validate the results obtained from the meta-analysis, we conducted RT-qPCR and western blot analyses on fibroblast cell lines and various PSC lines (Fig. 2b and c). Consistent with the findings of the meta-analysis, we observed TK2 expression exclusively in somatic cell lines, whereas it was absent in PSC lines. Next, to examine the expression of genes related to mtDNA replication and synthesis, we used the human secondary reprogramming cell line hiF-T. We evaluated the genes related to mtDNA synthesis and replication using mRNA sequencing data generated by Chicilla et al. during the reprogramming of hiF-T cells 27 . Notably, as reprogramming commenced, TK2 expression gradually decreased with the degree of reprogramming (Supplementary Fig. 1). To validate these results, we confirmed both TK2 mRNA and protein levels, mtDNA/nDNA ratio, and mitochondrial activity using hiF-T cells during somatic cell reprogramming (Fig. 2d–h). After DOX treatment, fully reprogrammed hiF-T cells showed a reduction in both TK2 mRNA and protein expression to baseline levels (day 30) (Fig. 2d and e). Furthermore, as the reprogramming process advanced, the mtDNA/nDNA ratio notably decreased, reaching a level of significance 30% lower than that of somatic cells after a complete 30-d reprogramming (Fig. 2f). Consistent with the reduction in TK2 expression and mtDNA copy numbers observed during reprogramming, a concurrent decline in overall mitochondrial activity was noted (Fig. 2g and h). Basal respiration considerably decreased from reprogramming day 20, and a declining trend in maximal respiration, ATP production, and spare respiration capacity was observed as reprogramming progressed. All mitochondrial metabolic parameters were markedly reduced after 30 days of complete reprogramming. Notably, proton leakage markedly decreased after day 10 of reprogramming (Fig. 2h). These results suggest that, apart from the reduction in oxidative phosphorylation processes within the mitochondria, there is also a decrease in the electrochemical gradient across the mitochondrial inner membrane during somatic cell reprogramming. This indicates that as somatic cells undergo reprogramming to attain pluripotency, there is a concurrent decrease in both mtDNA copy number and mitochondrial function. To investigate whether the expression of TK2 increases in differentiating cells, we examined TK2 expression in spontaneously differentiated EBs derived from PSCs. We observed a time-dependent increase in TK2 expression in the spontaneously differentiated EBs (Supplementary Fig. 2a and b). We observed distinct changes in TK2 expression between somatic cells and PSCs, indicating the need to investigate whether differences in TK2 expression can determine metabolic variations between these two cell types. Additionally, as the spontaneous differentiation of EB progressed, the mtDNA/nDNA ratio increased. Specifically, compared to undifferentiated iPSC, the mtDNA/nDNA ratio of EB differentiated for 15 d showed a marked increase of more than two-fold (Supplementary Fig. 2c). Consistent with the results of TK2 expression and the mtDNA/nDNA ratio reduction during spontaneous EB differentiation, an overall increase in mitochondrial activity was observed (Supplementary Fig. 2d and e). In particular, there was a considerable increase in maximal respiration, ATP production, and spare respiration capacity on the 15th day of spontaneous EB differentiation (Supplementary Fig. 2d and e). We observed distinct differences in TK2 expression between somatic cells and PSCs, indicating the need to investigate whether differences in TK2 expression could determine metabolic variations. TK2 inhibition induces mitochondrial functional changes To investigate whether TK2 expression directly regulates mitochondrial metabolic transition, we treated fibroblasts with TK2 inhibitors and assessed the mitochondrial functional changes. BJ1 fibroblasts were cultured for 72 h with various concentrations of the TK2 inhibitor AZT to examine its impact on TK2 expression. Protein expression of TK2 decreased in a dose-dependent manner, and the mtDNA/nDNA ratio markedly decreased (Fig. 3a and b). We examined the effects of AZT-induced mtDNA copy number reduction on protein expression in five OXPHOS complexes within the mitochondria. AZT reduced the expression of all five OXPHOS complexes, with no significant changes observed among them (Fig. 3c). Furthermore, to understand the influence of AZT treatment on cellular metabolic changes resulting from reduced mtDNA copy numbers and OXPHOS complex reduction, we measured the OCR after AZT treatment (Fig. 3d and e). AZT treatment decreased the overall OCR levels, including five respiratory parameters. These results suggest that reduced mtDNA copy number through TK2 inhibition can lead to a transition in the functional characteristics of mitochondria from those found in somatic cells to those seen in PSCs. Exploring the regulatory role of p53 and SIRT1 in TK2 expression during reprogramming The fact that decreased TK2 reduces the mtDNA copy number during the reprogramming process and contributes to the transition of cellular metabolism underscores the importance of exploring TK2 as a regulator of reprogramming. To identify potential upstream regulators of TK2 during the reprogramming process, we examined the genes that could bind to the TK2 promoter using publicly available databases (ChIPBase v2.0) 28 , with particular attention to the p53 gene. The p53 gene is not only associated with cell cycle-related genes, but also a well-known gene that can fine-tune the stemness of PSC cells. Furthermore, we are particularly interested in p53 as a potential upstream regulator of TK2 because it TK2 expression and mtDNA replication decrease in p53 knockout or deficient patients 26,29,30 . This suggests a relationship between reduced TK2 expression, decreased mtDNA replication, and loss of p53 function 22,31 . We first confirmed the expression of genes related to p53 proteins in fibroblasts, PSC, and reprogrammed hiF-T cells using western blotting. The secondary reprogramming cell line hiF-T showed a progressive increase in NANOG expression upon doxycycline treatment, indicating reprogramming was indeed occurring (Fig. 4a). In contrast to NANOG treatment, TK2 expression decreased in a time-dependent manner. Notably, while the overall protein levels of p53 remain relatively constant across all cells, the acceleration of reprogramming was associated with a reduction in p53 acetylation (Lys 382). This decrease in p53 acetylation was sustained at a low level even in iPSC cell lines. Notably, diminished acetylation of p53 correlates with a concurrent decrease in the expression of its downstream effector p21. The deacetylase SIRT1 exhibited a pattern of expression during reprogramming that was negatively correlated with the levels of p53 acetylation (Fig. 4a). To confirm whether p53 directly regulates the expression of TK2 during the reprogramming process, we performed ChIP analysis and confirmed that p53 indeed binds to the TK2 promoter. Compared to fibroblasts, PSCs exhibited a notable decrease in p53 binding affinity toward both p21 and the TK2 promoter regions (Fig. 4b and Supplementary Fig. 3). Subsequently, we treated the cells with sirtinol, a SIRT1 inhibitor, to assess the impact of SIRT1 reduction on TK2 expression and p53 activity (Fig. 4c). Sirtinol treatment increased the expression levels of both TK2 and p21. The increased expression of p21 suggests that SIRT1 enhances the activity of p53, confirming that the SIRT1-p53 axis is an upstream regulator of TK2 expression. mtDNA depletion induced by the SIRT1-TK2 axis increases somatic cell reprogramming We investigated whether SIRT1, an upstream regulator of TK2, directly modulates reprogramming through mtDNA depletion in hiF-T cells. To quantitatively assess the impact of SIRT1 regulation on reprogramming efficiency, we induced reprogramming of hiF-T cells by DOX treatment and conducted FACS analysis. Sirtinol treatment confirmed a reduction of SSEA-4/TRA1-60 double-positive cells by about 30% in sirtinol 15 uM (Fig. 5a and b), whereas SRT1720 treatment during hiF-T cell reprogramming resulted in an approximate 27% increase in reprogramming efficiency (Fig. 5a and b). Treatment with sirtinol increased p53 and TK2 expression and decreased NANOG expression in reprogrammed hiF-T cells for 20 d. Conversely, treatment with the SIRT1 activator SRT1720 reduced p53 and TK2 levels and increased NANOG expression (Fig. 5c). SIRT1 activity positively correlated with mtDNA expression (Fig. 5d). This indicates that the SIRT1-p53 axis can directly regulate the metabolic transition of reprogrammed cells, enhancing reprogramming efficiency. Discussion This study revealed that for somatic cells to acquire and maintain pluripotency while undergoing stochastic processes, a decrease in mtDNA copy number is crucial for the transition from OXPHOS-driven metabolism to aerobic glycolysis, with a key factor being the downregulation of the TK2 gene involved in mitochondrial thymidine biosynthesis. The TK enzyme, responsible for converting thymidine into its monophosphate form (dTMP; essential for DNA synthesis and repair), was first identified in the 1960s as an enzyme that adds a 5' phosphate to thymidine-deoxyribose 32 . Subsequently, two isoforms of TK, namely TK1 and TK2, were identified. TK1 is located on chromosome 17 (17q25.3), is highly proliferative, and plays a role in nuclear DNA synthesis, particularly in rapidly dividing fetal and tumor tissues 32 . In contrast, TK2, located on chromosome 16 (16q21), is responsible for mtDNA replication and is independent of cell cycle regulation 32 . Of particular interest was the observation that TK1 transcription and translated protein levels remained consistent, whereas TK2 showed differences (Supplementary Fig. 4a and b). To further investigate the protein synthesis of TK1 and TK2 based on cell characteristics, we treated fibroblasts with nocodazole to inhibit the cell cycle, followed by the analysis of TK1 and TK2 protein expression (Supplementary Fig. 4c). The results of our analysis showed that cell cycle inhibition by nocodazole treatment in fibroblasts (BJ1) markedly reduced the protein expression of TK1, but did not affect TK2 expression. From these data, we know that the lower expression of TK1 in somatic cells than in PSCs can be attributed, in part, to the relative differences in the cell cycle between the two cell types. However, unlike TK1, TK2 is expressed independent of the cell cycle, indicating its involvement in mtDNA synthesis regardless of the cell cycle stage. While TK2 expression is cell cycle-independent, the lower expression of TK2 in PSCs, despite their vigorous cell cycle, is likely due to the metabolic shift toward pluripotency maintenance and a preference for nucleic acid synthesis through the pentose phosphate pathway, which is derived from the metabolic transition to aerobic glycolysis. Based on our findings, we recognized the significance of reduced TK2 expression as a factor that regulates metabolic transitions during the reprogramming process. To gain a deeper understanding of stochastic reprogramming, we sought to identify upstream regulators of TK2. By analyzing publicly available literature, we confirmed that p53 directly binds to the TK2 promoter, thereby exerting its control. The importance of p53 expression and acetylation in the reprogramming and maintenance of pluripotency is well known 33 , 34 , 35 , 36 . SIRT1, a member of an extensive family of histone deacetylases, regulates p53 activity, influences the fate of numerous cells, and maintains homeostasis. One reason for the extremely low reprogramming efficiency is that all reprogramming factors are associated with the cell cycle, particularly c-Myc and klf4, which are potent oncogenic proteins, leading to the potential activation of p53 during reprogramming 37 . Activated p53 halts the cell cycle, induces cell death, and promotes aging, thereby inhibiting cell reprogramming. Consequently, the regulation of p53 activity during reprogramming is a major bottleneck in reprogramming technology. As a regulator capable of controlling p53 activity from an upstream position, SIRT1 plays a key role in maintaining genomic stability and inducing metabolic transitions during the reprogramming process. Specifically, SIRT1 is directly transcribed by OCT4; therefore, ectopically expressed OCT4 increases the expression of SIRT1, reducing the acetylation of p53. It is predicted that the decrease in the number of mtDNA copies and expression of TK2 during somatic cell reprogramming is a consequence of OCT4 activity 33 , 34 . Initially, it was expected that TK2 inhibition would improve the reprogramming efficiency, as it was reported to induce the Warburg effect and mitochondrial function. However, there is a potential for adverse effects on somatic cell reprogramming processes when directly blocking TK2 expression. Tk2 mutant mice (Tk2(KI/KI)) and conditions associated with mtDNA depletion exhibit high AMPK activity [28]. Moreover, AMPK acts as a metabolic barrier, inhibiting somatic cell reprogramming [29]. During somatic cell reprogramming by OSKM, decreased mitochondrial function leads to reduced ATP levels, which may trigger an energy homeostatic response mediated by AMPK. This suggests that energy metabolism and homeostatic mechanisms negatively affect the acquisition of pluripotency during cell reprogramming. Consequently, the notable reduction in TK2 postulates with the utilization of AZT does not significantly contribute to enhancing reprogramming efficiency (results not presented). This suggests that a full reduction in TK2 may induce metabolic stress, DNA damage, and replication stress during the reprogramming process, potentially negatively affecting the acquisition of pluripotency. The primary challenge to investigating alterations in reprogramming efficacy with AZT lies in the possible adverse impacts of directly inhibiting TK2 expression during somatic cell reprogramming. Given the intricacy of cellular mechanisms, the administration of AZT may lead to unforeseen repercussions or off-target effects linked to TK2 suppression, potentially influencing the overarching outcomes. However, iPSCs can also be successfully established in TK2 mutations fibroblasts 38 . It cannot be ruled out that these results are predominantly the result of cells that completed metabolic adaptation after TK2 knockout. Therefore, direct inhibition of TK2 expression should be carefully fine-tuned in the context of somatic cell reprogramming. Conclusion Our study sheds new light on metabolic transition as a crucial element in the regulation of mtDNA copy numbers during the transition from OXPHOS to aerobic glycolysis, overcoming one of the major barriers to dedifferentiation. Furthermore, we revealed, for the first time, that the SIRT1-p53 axis plays a pivotal role in lowering TK2 expression, impacting metabolic transitions, and the acquisition of pluripotency. Declarations CONFLICT OF INTEREST The authors declare no conflicts of interest. FUNDING This study was supported by the National Research Foundation of Korea (NRF-2020R1A6A3A13077021, RS-2023-00220207) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (KFRM-2022-00070557). This research was supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (24-BR-02-04). Chung-Ang University Graduate Research Scholarship (Academic Scholarship for College of Biotechnology and Natural Resources) in 2023. ACKNOWLEDGMENTS We express our sincere gratitude to Davide Cacciarelli from the Broad Institute for generously providing the hiF-T cell line used in this study for human reprogramming research. We would also like to thank the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute for their assistance with microscopy. Special thanks go to STEMOPIA. DATA AVAILABILITY Supplementary information accompanies the manuscript on the Experimental & Molecular Medicine website ( http://www.nature.com/emm/ ). References Hanna, J. et al. Direct Cell Reprogramming Is a Stochastic Process Amenable to Acceleration. Nature. 462, 595–601 (2009). Yamanaka, S. Elite and Stochastic Models for Induced Pluripotent Stem Cell Generation. Nature. 460, 49–52 (2009). Takahashi, K. & Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 126, 663–676 (2006). Takahashi, K. et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 131, 861–872 (2007). Orkin, S. H. & Hochedlinger, K. Chromatin Connections to Pluripotency and Cellular Reprogramming. Cell. 145, 835–50 (2011). Boyer, L. A. et al. Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells. Cell. 122, 947–56 (2005). Ishida, T., Nakao, S., Ueyama, T., Harada, Y. & Kawamura, T. Metabolic Remodeling During Somatic Cell Reprogramming to Induced Pluripotent Stem Cells: Involvement of Hypoxia-Inducible Factor 1. Inflamm Regen. 40, 8 (2020). Panopoulos, A. D. et al. The Metabolome of Induced Pluripotent Stem Cells Reveals Metabolic Changes Occurring in Somatic Cell Reprogramming. Cell Res. 22, 168–77 (2012). Varum, S. et al. Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts. PLoS One. 6, e20914 (2011). Prigione, A., Fauler, B., Lurz, R., Lehrach, H. & Adjaye, J. The Senescence-Related Mitochondrial/Oxidative Stress Pathway Is Repressed in Human Induced Pluripotent Stem Cells. Stem Cells . 28, 721 – 33 (2010). Folmes, C. D. et al. Somatic Oxidative Bioenergetics Transitions into Pluripotency-Dependent Glycolysis to Facilitate Nuclear Reprogramming. Cell Metab . 14, 264 – 71 (2011). Mathieu, J. et al. Hypoxia-Inducible Factors Have Distinct and Stage-Specific Roles During Reprogramming of Human Cells to Pluripotency. Cell Stem Cell. 14, 592–605 (2014). Prigione, A. et al. Hif1α Modulates Cell Fate Reprogramming through Early Glycolytic Shift and Upregulation of Pdk1-3 and Pkm2. Stem Cells . 32, 364 – 76 (2014). Hansson, J. et al. Highly Coordinated Proteome Dynamics During Reprogramming of Somatic Cells to Pluripotency. Cell Rep. 2, 1579–92 (2012). Vazquez-Martin, A. et al. Mitochondrial Fusion by Pharmacological Manipulation Impedes Somatic Cell Reprogramming to Pluripotency: New Insight into the Role of Mitophagy in Cell Stemness. Aging (Albany NY). 4, 393–401 (2012). Bukowiecki, R., Adjaye, J. & Prigione, A. Mitochondrial Function in Pluripotent Stem Cells and Cellular Reprogramming. Gerontology. 60, 174–82 (2014). Son, M. J. et al. Mitofusins Deficiency Elicits Mitochondrial Metabolic Reprogramming to Pluripotency. Cell Death Differ. 22, 1957–69 (2015). Prieto, J. et al. Early Erk1/2 Activation Promotes Drp1-Dependent Mitochondrial Fission Necessary for Cell Reprogramming. Nat Commun. 7, 11124 (2016). Folmes, C. D., Nelson, T. J. & Terzic, A. Energy Metabolism in Nuclear Reprogramming. Biomark Med . 5, 715 – 29 (2011). Suhr, S. T. et al. Mitochondrial Rejuvenation after Induced Pluripotency. PLoS One. 5, e14095 (2010). Wang, X. M. et al. Induced Pluripotent Stem Cell Models of Zellweger Spectrum Disorder Show Impaired Peroxisome Assembly and Cell Type-Specific Lipid Abnormalities. Stem Cell Res Ther. 6, 158 (2015). Armstrong, L. et al. Human Induced Pluripotent Stem Cell Lines Show Stress Defense Mechanisms and Mitochondrial Regulation Similar to Those of Human Embryonic Stem Cells. Stem Cells. 28, 661–673 (2010). Belogrudov, G. & Hatefi, Y. Catalytic Sector of Complex I (Nadh:Ubiquinone Oxidoreductase): Subunit Stoichiometry and Substrate-Induced Conformation Changes. Biochemistry. 33, 4571–6 (1994). Chan, E. M. et al. Live Cell Imaging Distinguishes Bona Fide Human Ips Cells from Partially Reprogrammed Cells. Nature Biotechnology. 27, 1033–1037 (2009). Sun, X. & St John, J. C. Modulation of Mitochondrial DNA Copy Number in a Model of Glioblastoma Induces Changes to DNA Methylation and Gene Expression of the Nuclear Genome in Tumours. Epigenetics & Chromatin. 11, 53 (2018). Bartesaghi, S. et al. Inhibition of Oxidative Metabolism Leads to P53 Genetic Inactivation and Transformation in Neural Stem Cells. Proc Natl Acad Sci U S A. 112, 1059–64 (2015). Cacchiarelli, D. et al. Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency. Cell. 162, 412–424 (2015). Zhou, K. R. et al. Chipbase V2.0: Decoding Transcriptional Regulatory Networks of Non-Coding Rnas and Protein-Coding Genes from Chip-Seq Data. Nucleic Acids Res. 45, D43-D50 (2017). Lebedeva, M. A., Eaton, J. S. & Shadel, G. S. Loss of P53 Causes Mitochondrial DNA Depletion and Altered Mitochondrial Reactive Oxygen Species Homeostasis. Biochimica et Biophysica Acta (BBA) - Bioenergetics . 1787, 328–334 (2009). Radivoyevitch, T. et al. Dntp Supply Gene Expression Patterns after P53 Loss. Cancers (Basel). 4, 1212–24 (2012). Lonergan, T., Bavister, B. & Brenner, C. Mitochondria in Stem Cells. Mitochondrion. 7, 289–296 (2007). Bitter, E. E., Townsend, M. H., Erickson, R., Allen, C. & O’Neill, K. L. Thymidine Kinase 1 through the Ages: A Comprehensive Review. Cell & Bioscience. 10, 138 (2020). Zhang, Z. N., Chung, S. K., Xu, Z. & Xu, Y. Oct4 Maintains the Pluripotency of Human Embryonic Stem Cells by Inactivating P53 through Sirt1-Mediated Deacetylation. Stem Cells. 32, 157–65 (2014). Lee, Y. L. et al. Sirtuin 1 Facilitates Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts through the Mir-34a and P53 Pathways. PLoS One. 7, e45633 (2012). Jain, A. K. et al. P53 Regulates Cell Cycle and Micrornas to Promote Differentiation of Human Embryonic Stem Cells. PLoS Biol. 10, e1001268 (2012). Lin, T. et al. P53 Induces Differentiation of Mouse Embryonic Stem Cells by Suppressing Nanog Expression. Nat Cell Biol. 7, 165–71 (2005). Fu, X., Wu, S., Li, B., Xu, Y. & Liu, J. Functions of P53 in Pluripotent Stem Cells. Protein Cell. 11, 71–78 (2020). Hernandez-Ainsa, C. et al. Generation of an Induced Pluripotent Stem Cell Line from a Compound Heterozygous Patient in Tk2 Gene. Stem Cell Res. 59, 102632 (2022). Additional Declarations (Not answered) Supplementary Files SupplementaryMaterialFiguresandTables.docx Cite Share Download PDF Status: Published Journal Publication published 26 Jun, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 29 Nov, 2024 Review # 3 received at journal 23 Nov, 2024 Reviewer # 3 agreed at journal 23 Nov, 2024 Review # 1 received at journal 15 Nov, 2024 Review # 2 received at journal 13 Nov, 2024 Reviewer # 2 agreed at journal 31 Oct, 2024 Reviewer # 1 agreed at journal 29 Oct, 2024 Reviewers invited by journal 28 Oct, 2024 Submission checks completed at journal 10 Oct, 2024 First submitted to journal 10 Oct, 2024 Unknown event 25 Sep, 2024 Editor assigned by journal 25 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5148938","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":371490782,"identity":"bcf21b27-3184-4dc6-9ba2-67c8ed58a819","order_by":0,"name":"Man Ryul Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPghlw8AgAWYkENbCBqHSSNdymBQt7M1HN/zccd6ef3YD44cfDGn5hLXwHEu72XvmduKMOweYJXsYciwbCGqRyDG7wdt2O8FAIoFBmoGhwoCwLfLvv93823bOHqiF+TdxWiR42G7zth1g3CCRwAa0JYcILTxpZrdl25ITZ9xIbLPsMUgjrIWf/fCzm2/b7Oz5ZyQfvvGjIpmwFiTA2MDAQJKGUTAKRsEoGAU4AQDd7DXKuWVVsgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2351-9679","institution":"Soonchunhyang Institute of Medi-bio Science (SIMS) and Institute of Tissue Regeneration, Soon Chun Hyang University, Cheonan-si, Chungcheongnam-do, Republic of Korea","correspondingAuthor":true,"prefix":"","firstName":"Man","middleName":"Ryul","lastName":"Lee","suffix":""},{"id":371490783,"identity":"d97b9a79-3936-4cf2-813c-6ccf70b1d48f","order_by":1,"name":"Hyun Kyu Kim","email":"","orcid":"","institution":"Soon Chun Hyang University","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Kyu","lastName":"Kim","suffix":""},{"id":371490784,"identity":"c2161a24-7127-4fcb-a17d-c65cad81f09c","order_by":2,"name":"Yena Song","email":"","orcid":"","institution":"Soonchunhyang Institute of Medi-bio Science (SIMS)","correspondingAuthor":false,"prefix":"","firstName":"Yena","middleName":"","lastName":"Song","suffix":""},{"id":371490785,"identity":"f8616dbe-55ec-4988-8d22-b443d0c37891","order_by":3,"name":"Minji Kye","email":"","orcid":"","institution":"Soonchunhyang Institute of Medi-bio Science (SIMS)","correspondingAuthor":false,"prefix":"","firstName":"Minji","middleName":"","lastName":"Kye","suffix":""},{"id":371490786,"identity":"c2b1254d-1641-48aa-9d5e-d4f516c11d2a","order_by":4,"name":"Byeongho Yu","email":"","orcid":"","institution":"Soonchunhyang Institute of Medi-bio Science (SIMS)","correspondingAuthor":false,"prefix":"","firstName":"Byeongho","middleName":"","lastName":"Yu","suffix":""},{"id":371490787,"identity":"f729b2ff-936c-4d5c-86ac-dd7f02395d2e","order_by":5,"name":"Hyung Kyu Choi","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Hyung","middleName":"Kyu","lastName":"Choi","suffix":""},{"id":371490788,"identity":"43dfb8eb-b702-4320-adbe-27992e687300","order_by":6,"name":"Sung-Hwan Moon","email":"","orcid":"","institution":"T\u0026R Biofab Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Sung-Hwan","middleName":"","lastName":"Moon","suffix":""}],"badges":[],"createdAt":"2024-09-25 05:00:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5148938/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5148938/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-025-01476-3","type":"published","date":"2025-06-26T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68418112,"identity":"39268e59-07a3-4d80-ae36-50f2a5b546bd","added_by":"auto","created_at":"2024-11-07 05:38:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21421831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial function and morphology characterization in somatic cells and pluripotent stem cells (PSCs).\u003c/strong\u003e (a) Phase-contrast microscopy (PCM) images depicting fibroblasts (MRC5, scale bar: 50 μm) and PSCs (hiPSC, H9-hESC, scale bar: 100 μm). Transmission electron microscopy (TEM) images of fibroblasts (MRC5) and PSCs (hiPSC, H9-hESC). Scale bar: 1 μm. Red arrows indicate mitochondria. (b) Mito stress test conducted on somatic cells and PSCs. Profiles presenting Mito stress test data for oxygen consumption rate (OCR) and calculated values for respiratory parameters. Results represent means ± SD (n=3). (c) Assessment of the reliance on respiration or glycolysis in fibroblasts and PSCs, illustrated by the plotted OCR/extracellular acidification rate (ECAR) ratio. Results represent means ± SD (n=6). (d) Mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratio in fibroblasts and PSCs. Results represent means ± SD (n=3). * denotes statistical significance (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0005, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.00005). ns: non-significant. Significant differences are assessed using Student's t-test or one-way ANOVA and Tukey's multiple comparison test. Statistical analyses are performed using GRAPHPAD 8.0.1\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/d5df51f036cdb80537f0b449.png"},{"id":68417575,"identity":"77b42382-aaae-442a-b90a-37bdb8bd1fe1","added_by":"auto","created_at":"2024-11-07 05:30:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5822659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDown-regulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTK2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene expression and mtDNA copy number as a molecular signature of human pluripotency.\u003c/strong\u003e (a) Violin plot illustrating log2 normalized read counts of mtDNA synthesis and replication-related genes in normal somatic cell lines (n = 78) and (n = 29), respectively; hiPSC lines (n = 51); and hESC lines derived from an RNA-seq database search. Results represent means ± SD. (b) RT-qPCR analysis depicting TK2 gene expression in somatic cells and PSCs. (c) Western blot analysis presenting TK2 protein expression in somatic cells and PSCs. (d) RT-qPCR analysis of \u003cem\u003eTK2, OCT4,\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e gene expression patterns at 10, 20, and 30 d during the reprogramming process. (e) Western blot analysis revealing TK2, NANOG, and GAPDH proteins expression patterns at 10, 20, and 30 d during reprogramming. (f) Changes in mtDNA/nDNA ratio during reprogramming. (g, h) Alterations in Mito stress test profile and respiratory parameters during reprogramming. Results represent means ± SD (n=3). * denotes statistical significance (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0005, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.00005). Significant differences are assessed through one-way ANOVA and Tukey's multiple comparison test. Statistical analyses are performed using GRAPHPAD 8.0.1. hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; PSC, pluripotent stem cell\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/94b3d0a0da2c8133982713e6.png"},{"id":68418104,"identity":"94992d1d-9861-4b0f-b3dc-97f1791036cf","added_by":"auto","created_at":"2024-11-07 05:38:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3651499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effect of TK2 in fibroblasts. \u003c/strong\u003e(a) Western blot analysis of TK2 expression after 72-h AZT treatment in fibroblasts (BJ1), where AZT (3'-azido-3'-deoxythymidine) serves as a TK2 inhibitor. (b) Alterations in mtDNA/nDNA ratio following AZT treatment in fibroblasts (BJ1). Results are expressed as means ± SD (n=3). (c) Changes in mitochondrial oxidative phosphorylation (OXPHOS) complex after AZT treatment in fibroblasts (BJ1). (d, e) Modulations in Mito stress test profile and respiratory parameters after a 72-h AZT 50 µM treatment in fibroblasts (BJ1). Results represent means ± SD (n=4). * denotes statistical significance (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Significant differences are analyzed using Student's t-test. Statistical analyses are conducted using GRAPHPAD 8.0.1. hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; PSC, pluripotent stem cell\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/fd640593a72494d141297275.png"},{"id":68417569,"identity":"e39ac0ad-9554-42d8-925c-f35e503a684a","added_by":"auto","created_at":"2024-11-07 05:30:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2258561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep53 directly participates in TK2 transcription.\u003c/strong\u003e (a) Expression patterns of SIRT1, p53 acetylation (Lys 382), p21, TK2, and NANOG during somatic cell reprogramming. (b) ChIP-qPCR analysis illustrating the binding activity of p53 to the TK2 promoter in fibroblasts and PSCs. (c) RT-qPCR analysis of p21 and TK2 after 24 h of sirtinol (SIRT1 inhibitor) treatment in PSCs. Results represent means ± SD (n=3). * denotes statistical significance (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Significant differences are assessed through Student's t-test or one-way ANOVA and Tukey's multiple comparison test. Statistical analyses are conducted using GRAPHPAD 8.0.1.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/1890970247dfbedd396298ad.png"},{"id":68417571,"identity":"51f37209-cf68-431e-9c88-84753edfb1f9","added_by":"auto","created_at":"2024-11-07 05:30:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2969597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of somatic cell reprogramming efficiency with SIRT1 inhibitor (sirtinol) and SIRT1 activator (SRT1720).\u003c/strong\u003e (a) Flow cytometry analysis of the surface markers TRA-1-60 (FITC) and SSEA4 (APC) in sirtinol and SRT1720 treated reprogrammed cells. (b) Statistical analysis of somatic cell reprogramming efficiency based on FACS data. (c) Alterations in protein expression patterns of SIRT1, p53 acetylation, p21, TK2, and NANOG due to SIRT1 regulation during somatic cell reprogramming. (d) Changes in mtDNA/nDNA ratio attributed to SIRT1 regulation in somatic cell reprogramming. Results are presented as means ± SD (n=3). * denotes statistical significance. Significant differences are assessed through Student's t-test or one-way ANOVA and Tukey's multiple comparison test. Statistical analyses are conducted using GRAPHPAD 8.0.1. mtDNA, mitochondrial DNA; nDNA, nuclear DNA\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/583d1681a37c571eeeba1177.png"},{"id":85541990,"identity":"f3d9adc7-8352-4110-b79c-7f2577ea4d63","added_by":"auto","created_at":"2025-06-27 07:06:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34939635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/36dfdb3d-1cef-4948-acb8-2bae6da1e91c.pdf"},{"id":68417573,"identity":"637b879a-f8db-4a97-a28a-818c88c9d2d1","added_by":"auto","created_at":"2024-11-07 05:30:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1196806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFiguresandTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5148938/v1/2dfd71141a2930c1d5f4b01f.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Changes in mitochondrial thymidine metabolism and mtDNA copy number during induced pluripotency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInduced pluripotent stem cells (iPSCs) are reprogrammed cells obtained by introducing reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) into somatic cells, leading them through stochastic and determinate phases to acquire pluripotency \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Reprogramming orchestrates dynamic shifts in gene expression profiles, epigenetic landscapes, metabolic traits, and cellular morphology within somatic cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Despite comprehensive investigations, the acquisition of pluripotency from somatic cells during the stochastic phase is acknowledged as a sluggish and inefficient process owing to its intricate nature \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Endeavors to illuminate the intricacies of reprogramming persist; however, the underlying metabolic mechanisms pivotal for acquiring pluripotency remain largely unknown.\u003c/p\u003e \u003cp\u003eTo ensure an essential energy supply for cell survival, cells typically break down glucose into pyruvate in the presence of oxygen, subsequently subjecting it to oxidative phosphorylation (OXPHOS) within the mitochondria to synthesize ATP. However, even in the presence of sufficient oxygen, pluripotent stem cells (PSCs) engage in aerobic glycolysis to convert pyruvate into lactate instead of transporting pyruvate to the mitochondria for ATP production. This process is relatively inefficient from an energy production perspective. However, in cases where the metabolic flux is sufficiently high, and during rapid cellular proliferation that characterizes undifferentiated embryonic stem cells (ESCs) and iPSCs, aerobic glycolysis can be considered a favorable metabolic mechanism \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Aerobic glycolysis can produce a rapid supply of ATP and is recognized as a pivotal process that provides essential building blocks, including nucleic acids generated through the pentose phosphate pathway, amino acids, and lipids required for biosynthesis, particularly during the observed robust proliferation at the initial stages. Therefore, the process by which somatic cells acquire pluripotency through reprogramming involves an essential concomitant transition from the predominantly occurring somatic cell metabolism, characterized by OXPHOS, to the metabolic profile characteristic of PSCs, namely, aerobic glycolysis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. During reprogramming, it has been reported that the fundamental mechanism behind metabolic changes involves the upregulation of glycolytic genes, contributing to the activation of this process, while concurrently downregulating mitochondrial respiratory chain complex genes to enhance reprogramming efficiency \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, morphological alterations in the mitochondria influence reprogramming efficiency \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. During reprogramming, mature tubular and cristae-rich mitochondria found in somatic cells transition into immature spherical mitochondria with reduced cristae as pluripotency is acquired \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These morphological and functional changes in mitochondria are closely associated with the acquisition of metabolic traits in PSCs and directly affect reprogramming efficiency \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably, PSCs, characterized by aerobic glycolysis, exhibit a marked decrease in mitochondrial DNA (mtDNA) copy numbers compared with that in somatic cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. mtDNA, a circular DNA molecule spanning 16.6 kb, encodes 13 essential protein subunits of mitochondrial respiratory chain complexes I, III, IV, and V, as well as 22 tRNAs and 2 rRNAs required for the translation of mitochondrial subunits \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, changes in the mtDNA copy number can directly affect mitochondrial function and the acquisition of pluripotency during reprogramming. The regulation of mtDNA copy number is tightly controlled by nuclear-encoded mtDNA replication factors \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Loss of function of genes involved in mtDNA replication and synthesis leads to decreased mtDNA copy number, mitochondrial defects, and increased reliance on aerobic glycolysis, resembling the Warburg effect \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Similarly, metabolic conversion from OXPHOS to glycolysis also occurs during somatic cell reprogramming and is accompanied by a decrease in mtDNA copy number \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Furthermore, human ESCs have a relatively low number of mtDNA copies compared with somatic cells \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Contrastingly, mitochondria undergo marked changes during cellular differentiation, leading to an increase in mtDNA copy number and a mature structural state characterized by a dense matrix, complex cristae, and dispersed localization in the cytoplasm \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This suggests that mtDNA copy number is closely linked to the identity of stem cells during induced pluripotency. However, the molecular mechanisms underlying alterations in mitochondrial function and mtDNA copy number are not well understood. In this study, we focus on the differential expression of thymidine kinase 2 (TK2) in somatic cells and pluripotent stem cells (PSCs), and its potential role in regulating mitochondrial DNA (mtDNA) copy number and the acquisition of pluripotency characteristics. Human cells possess two enzymes, TK1 and TK2, that are involved in phosphorylating thymidine, a building block of DNA necessary for DNA replication and synthesis. TK1 demonstrates cell cycle-dependent activity in the cytoplasm, showing high activity in rapidly dividing cells, including stem cells and tumor cells, whereas it is ubiquitinated and degraded in slow-dividing cells or cells with less active DNA replication \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In contrast, TK2, encoded by a gene located on chromosome 16, is translated and targeted to the mitochondria, where it functions independently of the cell cycle and is involved in mitochondrial thymidine synthesis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This distinction in the roles of TK1 and TK2 suggests a unique metabolic regulation in PSCs and their mitochondrial function.\u003c/p\u003e \u003cp\u003eThis study aims to explore the role of TK2 in mtDNA synthesis and its contribution to pluripotency. We intend to investigate the expression levels of TK2 during the reprogramming process and identify potential genetic regulators of TK2 that influence mtDNA copy number. These insights will help further our understanding of the metabolic characteristics that are essential for maintaining stemness and enhancing reprogramming efficiency in PSCs.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eCulture and maintenance of human\u003c/strong\u003e\u003cstrong\u003e ESCs (hESCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hESC cell lines, H1 and H9 (WiCell Research Institute, Madison, WI, USA), and the hiPSC cell lines CMC-hiPSC-003, 009, and 011 (Korea Centers for Disease Control and Prevention, Osong, Korea), were maintained in complete TeSR-E8 medium (StemCell Technologies, Vancouver, Canada) for feeder-free culture. PSCs were plated on vitronectin-coated dishes. The medium was replaced every 24 h. For passaging, the cells were enzymatically detached using TrypLE Express (Gibco, Carlsbad, CA, USA) and transferred to a new coated dish every 5 d. Passages were prepared at split ratios of 1:5 or 1:10 The fibroblast cell lines BJ1, MRC5, and NT2 (human embryonal carcinoma stem cells) were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM high glucose; Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (Corning) and 100\u0026times; penicillin-streptomycin (Corning). Fibroblasts and NT2 cells were routinely passaged every 4\u0026ndash;5 d using TrypLE Express. All cells were maintained in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEmbryoid body (\u003c/strong\u003e\u003cstrong\u003eEB) generation to induce spontaneous \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor EB generation, clumps of undifferentiated hESCs were mechanically detached using glass pipettes. Subsequently, these clumps were seeded onto non-adhesive bacterial dishes in differentiation medium devoid of basic fibroblast growth factors. The cells were then allowed to spontaneously aggregate to form EBs. The differentiation medium was refreshed on a daily basis, comprised of DMEM/F12 (Invitrogen, Gibco) supplemented with 20% KnockOut Serum Replacement (Invitrogen, Gibco), 1 mM glutamine, 1% nonessential amino acids, 0.1% penicillin/streptomycin, and 0.1 mM beta-mercaptoethanol.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMaintenance and reprogramming using secondary reprogramming system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehIF-T fibroblasts for somatic cell reprogramming (from Davide Cacchiarelli; Broad Institute, Cambridge, MA, USA) were cultured in an optimized DMEM/F12 culture medium supplemented with 10% fetal bovine serum \u003csup\u003e27\u003c/sup\u003e. The reprogramming was performed using the TeSR\u0026trade;-E7\u0026trade; Medium for Reprogramming (2-Component) containing Vitronectin XF and doxycycline hyclate (D9891; Sigma\u0026ndash;Aldrich, St Louis, MO, USA), according to the manufacturer\u0026rsquo;s instructions. Primosin (InvivoGen; ant-pm-1) at 50 \u0026mu;g/mL was used to prevent microbial contamination during somatic cell reprogramming. hIF-T-derived iPSCs were cultured in TeSR\u0026trade;-E8\u0026trade; medium (STEMCELL Technologies; 05990) or StemMACS\u0026trade; iPS-Brew XF (Miltenyi Biotec; 130-104-368) containing Vitronectin XF.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eOptical microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphologies of the ESCs, iPSCs colonies, and fibroblasts were observed using an optical microscope (CKX53; Olympus, Tokyo, Japan). Cell images were captured using an eXcope X9 (Dixi Science, Daejeon, Korea).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eElectron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were cultured on a 100-mm culture dish, washed twice with PBS, and fixed with 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. The samples were lifted using a cell lifter (Corning), centrifuged (4,000 rpm, 4\u0026deg;C, 30 min) and stored at 4\u0026deg;C until further processing. The samples were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Eponate-12 resin (Ted Pella, Redding, CA, USA). One-micrometer-thick sections were prepared using a Reichert-Jung UltraCut E ultramicrotome (Reichert Technologies, Depew, NY, USA), stained with toluidine blue, and imaged (Olympus BX-51). Seventy-nanometer-thick sections per block were placed on formvar-coated slot grids, stained with uranyl acetate/lead citrate, and imaged using transmission electron microscopy (H-7600; Hitachi, Tokyo, Japan).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMito Stress Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer\u0026rsquo;s protocols. Fibroblasts were plated in wells of an XF96 cell culture microplate and incubated at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h to ensure attachment. PSCs were plated in wells of an XF96 cell culture microplate coated with vitronectin XF (STEMCELL Technologies; 07180) and incubated at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h to ensure attachment. Ten micrometers of Y-27632 was added to the culture medium only for the first 24 h after seeding. The assay was initiated after the cells were equilibrated for 1 h in XF assay medium supplemented with 10 mM glucose, 5 mM sodium pyruvate, and 2 mM glutamine in a non-CO\u003csub\u003e2\u003c/sub\u003e incubator. The substrate-based metabolic assay was performed by applying 10 mM glucose injections after starvation in XF DMEM (pH 7.4; Seahorse Bioscience). Mito Stress Test of the cells was employed before and after the sequential injection of 2 \u0026mu;M oligomycin, 1\u0026mu;M Carbonyl cyanide m‐chlorophenylhydrazone (CCCP), 0.5 \u0026mu;M rotenone, and 0.5 \u0026mu;M antimycin A. Each OCR and ECAR value was normalized after protein quantification using BCA Protein Assay Kit (Thermo Fisher Scientific, Wilmington, DE, USA; 23227) at the final step of the assay.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRNA extraction, reverse transcription, and quantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed with easy-BLUE (iNtRON, Daejeon, Korea) and total RNA was extracted. RNA was quantified using a NanoDrop spectrophotometer. One microgram of RNA was reverse-transcribed using the All-in-One 5\u0026times; First Strand cDNA Synthesis Master Mix kit (CellScript, Madison, WI, USA), and quantitative PCR was performed using TOPreal\u0026trade; qPCR 2X PreMIX (Enzynomics, Daejeon, Korea). The experiments were performed according to the manufacturer\u0026rsquo;s instructions. RNA was extracted from each treatment condition, and the experiments were performed in triplicate. Primer sequences were designed according to the Integrated DNA Technologies website (IDT; https://sg.idtdna.com/pages). All primers were validated for efficiency, and data were obtained from experiments performed in triplicate.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates were extracted using NP40 (Elpis Biotech, Daejeon, Korea) and a 100\u0026times; protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA;5872S). Cell lysates were centrifuged at 13,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C, and the supernatants were obtained. The total protein concentration in each supernatant was measured using a BCA Protein Assay (Thermo Fisher Scientific; 23227). The protein samples were separated by electrophoresis using 8\u0026ndash;12% SDS-polyacrylamide gels and transferred to 0.2-\u0026mu;M polyvinylidene fluoride blotting membranes (Amersham, Little Chalfont, UK). The membranes were blocked in 5% bovine serum albumin (BSA; BioShop, Burlington, Canada; ALB001.100) in TBS-T (50 mM Tris, 0.15 M sodium chloride, 0.05% Tween 20) for 1 h at 25\u0026deg;C. After blocking, the membranes were incubated with the primary antibody (1:1000) diluted in 1% BSA in TBS-T for 16 h at 4\u0026deg;C. The membranes were washed three times for 10 min with TBS-T at room temperature and then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2500) diluted in 1% BSA in TBS-T for 1 h at room temperature. Chemiluminescence was detected using a Pico EPD Western Blot Detection Kit (Elpis Biotech, EBP-1073) or Amersham ECL Prime Western Blotting Detection Reagent (Amersham, RPN2232). The antibodies used in this study are listed in Supplementary Table 1.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMeta-analysis of gene expression data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-Seq data related to somatic cells, PSCs, and reprogramming were collected and downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, STAR aligner software was used to map the data onto the GRCh38 reference genome. Following mapping, the obtained read counts were used to analyze the expression levels of the genes of interest. The data (access codes) used for the analysis are listed in Supplementary Table 2. The results were visualized using R version 4.1.1. The ggplot2 R package was used to create violin plots for visualization.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eReverse transcription quantitative PCR (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed with easy-BLUE (iNtRON), and total RNA was extracted. The RNA was quantified with a Nanodrop ND-2000 spectrometer (Thermo Fisher Scientific). One microgram of the RNA was reverse-transcribed using the cDNA Master Mix (CellSafe, Yongin, Korea; CDS-100), and quantitative PCR was performed using TOPreal\u0026trade; qPCR 2X PreMIX (Enzynomics;RT500M). cDNA synthesis and qPCR were performed according to manufacturer\u0026apos;s instructions. Primer sequences were designed using the PCR primer design tool IDT (https://sg.idtdna.com/pages). All primers were validated for efficiency and specificity, and experiments were performed in triplicates. The primer sequences are listed in Supplementary Table 3.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial DNA copy number analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell pellet was lysed with 500 \u0026mu;L lysis buffer (Tris-HCL, 2 M pH 9.0; EDTA, 0.1 M pH 8.0; 0.5% SDS; and distilled water). Then, 10 \u0026mu;L 20 mg/mL Proteinase K Solution (Invitrogen; 25530049) was added, followed by an incubation for 3 h at 55\u0026deg;C or until the cell pellet was completely dissolved. Subsequently, following a treatment with RNase A (Thermo Fisher Scientific; EN0531, 10 mg/mL) at 65\u0026deg;C for 1 h, total cellular DNA was isolated using a conventional phenol-chloroform DNA extraction method. The DNA was dissolved in Tris-EDTA (TE) buffer (10 mM Tris\u0026ndash;HCl and 1 mM EDTA, pH 8.0). The DNA from all samples was diluted to 50 ng. To quantify mtDNA copy number, the nuclear DNA/mtDNA ratio was determined by employing the \u003cem\u003eND1 \u003c/em\u003egene primer specific to mtDNA and the genomic DNA \u0026beta;-globin gene primer. The primer sequences are listed in Supplementary Table 4. Quantitative PCR was performed using TOPreal\u0026trade; qPCR 2X PreMIX (RT500M; Enzynomics). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP) and qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP analysis was conducted using a TruChIP Chromatin Shearing Kit (Covaris, Woburn, MA, USA; 520127). Initially, cells were subjected to a 3-min treatment with methanol-free 16% paraformaldehyde (Thermo Fisher Scientific; 28908) to enable fixation, followed by an isolation of the nuclei. Subsequently, the isolated nuclei were fragmented to approximately 500 bp using an next generation sequencing sample processor, M220 (Covaris). Immunoprecipitation was performed using the specific antibody and isotyped IgG, and the chromatin bound to the antibody was subsequently separated utilizing Dynabeads\u0026trade; Protein G (Invitrogen). Precipitated chromatin was treated with proteinase K (Invitrogen; 25530049, 20 mg/mL) and reverse cross-linked by heating at 65\u0026deg;C for 4\u0026ndash;5 h (or overnight). Then, after treatment with RNase A (Thermo Fisher Scientific; EN0531, 10 mg/mL) at 65\u0026deg;C for 1 h, DNA was purified using a conventional phenol-chloroform DNA extraction method. Finally, the target enrichment was assessed by quantitative analysis using qPCR. Information regarding the primer sequences used is provided in Supplementary Table 5.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of somatic cell reprogramming efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReprogramming samples were dissociated into individual cells using TrypLE\u0026trade; Express Enzyme (1X) without phenol red (Gibco\u0026trade;). After harvesting and PBS washing, single-cell suspensions were permeabilized and fixed in a 4% paraformaldehyde solution (in PBS). The cells were labeled with SSEA4 (APC) and TRA-1-60 (FITC) antibodies, followed by washing. The fluorescence-activated cell sorting (FACS) antibodies used in this study are listed in Supplementary Table 1. All steps involving cell permeabilization, fixation, staining, and data acquisition were performed on the same day using the same instrument for each experiment to maintain consistency. Analysis and cell sorting were performed using a FACS Canto II instrument from BD Biosciences (Franklin Lakes, NJ, USA), and the data were processed using BD FACSCanto\u0026trade; Clinical Software.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed three times in triplicate, and data are presented as mean \u0026plusmn; standard deviation (SD) for statistical comparison. Statistical significance of differences between groups was evaluated using Student\u0026rsquo;s t-test. \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, Inc., San Diego, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDown-regulation of \u003cem\u003eTK2\u003c/em\u003e gene expression and mtDNA copy number is a molecular signature of human pluripotency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the metabolic changes occurring during somatic cell reprogramming, we compared the morphological features of mitochondria between fibroblasts (MRC5) and PSCs (hiPSCs; CMC-003 and hESC; H1) using electron microscopy. The analysis revealed that fibroblasts displayed mitochondria with dense cristae and an elongated tubular shape, whereas PSCs exhibited mitochondria with fewer cristae and a round shape (Fig. 1a). To determine whether these morphological differences between fibroblasts and PSCs corresponded to functional disparities, we measured mitochondrial function and the OCR/ECAR ratio using Seahorse XF96 with a series of drug injections consisting of an ATP synthase inhibitor (oligomycin), an uncoupler (CCCP), and electron transport chain inhibitors (antimycin A and rotenone). Human PSCs displayed considerably lower overall OCR levels and OCR/ECAR ratios than fibroblasts (Fig. 1b and c). Specifically, during basal respiration or maximal respiration, the OCR in hPSCs was approximately 2\u0026ndash;5 times lower than that in fibroblasts (Fig. 1b). In hPSCs, OCR linked to mitochondrial inner membrane proton leakage was approximately 11-fold lower than that in fibroblasts, and OCR linked to ATP production was also considerably lower (Fig. 1b). This result indicates that mitochondrial function and ATP production efficiency of hPSCs are relatively low compared to those of fibroblasts. Additionally, hPSCs exhibited a markedly reduced spare-respiratory capacity (calculated as maximum OCR minus basal OCR) compared to fibroblasts (Fig. 1b), indicating that hPSCs maintain morphologically immature mitochondrial features as well as functionally immature characteristics. One particularly noteworthy observation was the marked difference in mtDNA copy numbers between fibroblasts and PSCs (Fig. 1d). mtDNA encodes 13 genes that are critical for OXPHOS. Maintaining a certain mtDNA copy number is essential for mitochondrial function. Therefore, it can be hypothesized that the metabolic shift during reprogramming may be attributed to changes in mtDNA copy numbers, resulting in decreased mitochondrial function and acquired pluripotency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of TK2 as a key regulator in mtDNA maintenance during reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify specific genes related to mtDNA replication and synthesis, we conducted a meta-analysis using open RNA-seq databases (Fig. 2a). RNA-seq data for somatic cells, iPSCs, ESCs, and reprogramming processes were downloaded from the GEO database (Supplementary Table 2). After data mapping and normalization using a standardized pipeline, we analyzed the expression patterns of the target genes.\u003c/p\u003e\n\u003cp\u003eThe constructed database accurately reflected the expression of pluripotency markers, such as OCT4, SOX2, and NANOG (Fig. 2a). Most genes involved in mtDNA synthesis and replication were considerably downregulated during reprogramming, with TK2 showing the most notable decrease (Fig. 2b, Supplementary Table 1). We propose that for mtDNA synthesis to occur efficiently and emerge as a pivotal factor in mitochondrial metabolic transition, thymidine supply, primarily governed by TK2, takes precedence over other factors, thereby underscoring the importance of TK2 in the regulation of mtDNA copy numbers. Therefore, we aimed to elucidate the functional characteristics of TK2 during reprogramming.\u003c/p\u003e\n\u003cp\u003eTo validate the results obtained from the meta-analysis, we conducted RT-qPCR and western blot analyses on fibroblast cell lines and various PSC lines (Fig. 2b and c). Consistent with the findings of the meta-analysis, we observed TK2 expression exclusively in somatic cell lines, whereas it was absent in PSC lines. Next, to examine the expression of genes related to mtDNA replication and synthesis, we used the human secondary reprogramming cell line hiF-T. We evaluated the genes related to mtDNA synthesis and replication using mRNA sequencing data generated by Chicilla et al. during the reprogramming of hiF-T cells \u003csup\u003e27\u003c/sup\u003e. Notably, as reprogramming commenced, TK2 expression gradually decreased with the degree of reprogramming (Supplementary Fig. 1). To validate these results, we confirmed both \u003cem\u003eTK2\u003c/em\u003e mRNA and protein levels, mtDNA/nDNA ratio, and mitochondrial activity using hiF-T cells during somatic cell reprogramming (Fig. 2d\u0026ndash;h). After DOX treatment, fully reprogrammed hiF-T cells showed a reduction in both \u003cem\u003eTK2\u003c/em\u003e mRNA and protein expression to baseline levels (day 30) (Fig. 2d and e).\u003c/p\u003e\n\u003cp\u003eFurthermore, as the reprogramming process advanced, the mtDNA/nDNA ratio notably decreased, reaching a level of significance 30% lower than that of somatic cells after a complete 30-d reprogramming (Fig. 2f). Consistent with the reduction in TK2 expression and mtDNA copy numbers observed during reprogramming, a concurrent decline in overall mitochondrial activity was noted (Fig. 2g and h). Basal respiration considerably decreased from reprogramming day 20, and a declining trend in maximal respiration, ATP production, and spare respiration capacity was observed as reprogramming progressed. All mitochondrial metabolic parameters were markedly reduced after 30 days of complete reprogramming. Notably, proton leakage markedly decreased after day 10 of reprogramming (Fig. 2h). These results suggest that, apart from the reduction in oxidative phosphorylation processes within the mitochondria, there is also a decrease in the electrochemical gradient across the mitochondrial inner membrane during somatic cell reprogramming. This indicates that as somatic cells undergo reprogramming to attain pluripotency, there is a concurrent decrease in both mtDNA copy number and mitochondrial function. To investigate whether the expression of TK2 increases in differentiating cells, we examined TK2 expression in spontaneously differentiated EBs derived from PSCs. We observed a time-dependent increase in TK2 expression in the spontaneously differentiated EBs (Supplementary Fig. 2a and b). We observed distinct changes in TK2 expression between somatic cells and PSCs, indicating the need to investigate whether differences in TK2 expression can determine metabolic variations between these two cell types. Additionally, as the spontaneous differentiation of EB progressed, the mtDNA/nDNA ratio increased. Specifically, compared to undifferentiated iPSC, the mtDNA/nDNA ratio of EB differentiated for 15 d showed a marked increase of more than two-fold (Supplementary Fig. 2c). Consistent with the results of TK2 expression and the mtDNA/nDNA ratio reduction during spontaneous EB differentiation, an overall increase in mitochondrial activity was observed (Supplementary Fig. 2d and e). In particular, there was a considerable increase in maximal respiration, ATP production, and spare respiration capacity on the 15th day of spontaneous EB differentiation (Supplementary Fig. 2d and e). We observed distinct differences in TK2 expression between somatic cells and PSCs, indicating the need to investigate whether differences in TK2 expression could determine metabolic variations.\u003cs\u003e\u003c/s\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTK2 inhibition induces mitochondrial functional changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether TK2 expression directly regulates mitochondrial metabolic transition, we treated fibroblasts with TK2 inhibitors and assessed the mitochondrial functional changes. BJ1 fibroblasts were cultured for 72 h with various concentrations of the TK2 inhibitor AZT to examine its impact on TK2 expression. Protein expression of TK2 decreased in a dose-dependent manner, and the mtDNA/nDNA ratio markedly decreased (Fig. 3a and b). We examined the effects of AZT-induced mtDNA copy number reduction on protein expression in five OXPHOS complexes within the mitochondria. AZT reduced the expression of all five OXPHOS complexes, with no significant changes observed among them (Fig. 3c). Furthermore, to understand the influence of AZT treatment on cellular metabolic changes resulting from reduced mtDNA copy numbers and OXPHOS complex reduction, we measured the OCR after AZT treatment (Fig. 3d and e). AZT treatment decreased the overall OCR levels, including five respiratory parameters. These results suggest that reduced mtDNA copy number through TK2 inhibition can lead to a transition in the functional characteristics of mitochondria from those found in somatic cells to those seen in PSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExploring the regulatory role of p53 and SIRT1 in TK2 expression during reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fact that decreased TK2 reduces the mtDNA copy number during the reprogramming process and contributes to the transition of cellular metabolism underscores the importance of exploring TK2 as a regulator of reprogramming. To identify potential upstream regulators of TK2 during the reprogramming process, we examined the genes that could bind to the TK2 promoter using publicly available databases (ChIPBase v2.0) \u003csup\u003e28\u003c/sup\u003e, with particular attention to the \u003cem\u003ep53\u003c/em\u003e gene. The p53 gene is not only associated with cell cycle-related genes, but also a well-known gene that can fine-tune the stemness of PSC cells. Furthermore, we are particularly interested in p53 as a potential upstream regulator of TK2 because it TK2 expression and mtDNA replication decrease in \u003cem\u003ep53\u0026nbsp;\u003c/em\u003eknockout or deficient patients \u003csup\u003e26,29,30\u003c/sup\u003e. This suggests a relationship between reduced TK2 expression, decreased mtDNA replication, and loss of p53 function \u003csup\u003e22,31\u003c/sup\u003e. We first confirmed the expression of genes related to p53 proteins in fibroblasts, PSC, and reprogrammed hiF-T cells using western blotting. The secondary reprogramming cell line hiF-T showed a progressive increase in NANOG expression upon doxycycline treatment, indicating reprogramming was indeed occurring (Fig. 4a). In contrast to NANOG treatment, TK2 expression decreased in a time-dependent manner. Notably, while the overall protein levels of p53 remain relatively constant across all cells, the acceleration of reprogramming was associated with a reduction in p53 acetylation (Lys 382). This decrease in p53 acetylation was sustained at a low level even in iPSC cell lines. Notably, diminished acetylation of p53 correlates with a concurrent decrease in the expression of its downstream effector p21. The deacetylase SIRT1 exhibited a pattern of expression during reprogramming that was negatively correlated with the levels of p53 acetylation (Fig. 4a). To confirm whether p53 directly regulates the expression of TK2 during the reprogramming process, we performed ChIP analysis and confirmed that p53 indeed binds to the TK2 promoter. Compared to fibroblasts, PSCs exhibited a notable decrease in p53 binding affinity toward both p21 and the TK2 promoter regions (Fig. 4b and Supplementary Fig. 3). Subsequently, we treated the cells with sirtinol, a SIRT1 inhibitor, to assess the impact of SIRT1 reduction on TK2 expression and p53 activity (Fig. 4c). Sirtinol treatment increased the expression levels of both TK2 and p21. The increased expression of p21 suggests that SIRT1 enhances the activity of p53, confirming that the SIRT1-p53 axis is an upstream regulator of TK2 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emtDNA depletion induced by the SIRT1-TK2 axis increases somatic cell reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether SIRT1, an upstream regulator of TK2, directly modulates reprogramming through mtDNA depletion in hiF-T cells. To quantitatively assess the impact of SIRT1 regulation on reprogramming efficiency, we induced reprogramming of hiF-T cells by DOX treatment and conducted FACS analysis. Sirtinol treatment confirmed a reduction of SSEA-4/TRA1-60 double-positive cells by about 30% in sirtinol 15 uM (Fig. 5a and b), whereas SRT1720 treatment during hiF-T cell reprogramming resulted in an approximate 27% increase in reprogramming efficiency (Fig. 5a and b). Treatment with sirtinol increased p53 and TK2 expression and decreased NANOG expression in reprogrammed hiF-T cells for 20 d. Conversely, treatment with the SIRT1 activator SRT1720 reduced p53 and TK2 levels and increased NANOG expression (Fig. 5c). SIRT1 activity positively correlated with mtDNA expression (Fig. 5d). This indicates that the SIRT1-p53 axis can directly regulate the metabolic transition of reprogrammed cells, enhancing reprogramming efficiency.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study revealed that for somatic cells to acquire and maintain pluripotency while undergoing stochastic processes, a decrease in mtDNA copy number is crucial for the transition from OXPHOS-driven metabolism to aerobic glycolysis, with a key factor being the downregulation of the \u003cem\u003eTK2\u003c/em\u003e gene involved in mitochondrial thymidine biosynthesis.\u003c/p\u003e \u003cp\u003eThe TK enzyme, responsible for converting thymidine into its monophosphate form (dTMP; essential for DNA synthesis and repair), was first identified in the 1960s as an enzyme that adds a 5' phosphate to thymidine-deoxyribose \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Subsequently, two isoforms of TK, namely TK1 and TK2, were identified. TK1 is located on chromosome 17 (17q25.3), is highly proliferative, and plays a role in nuclear DNA synthesis, particularly in rapidly dividing fetal and tumor tissues \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In contrast, TK2, located on chromosome 16 (16q21), is responsible for mtDNA replication and is independent of cell cycle regulation \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Of particular interest was the observation that TK1 transcription and translated protein levels remained consistent, whereas TK2 showed differences (Supplementary Fig.\u0026nbsp;4a and b). To further investigate the protein synthesis of TK1 and TK2 based on cell characteristics, we treated fibroblasts with nocodazole to inhibit the cell cycle, followed by the analysis of TK1 and TK2 protein expression (Supplementary Fig.\u0026nbsp;4c). The results of our analysis showed that cell cycle inhibition by nocodazole treatment in fibroblasts (BJ1) markedly reduced the protein expression of TK1, but did not affect TK2 expression. From these data, we know that the lower expression of TK1 in somatic cells than in PSCs can be attributed, in part, to the relative differences in the cell cycle between the two cell types. However, unlike TK1, TK2 is expressed independent of the cell cycle, indicating its involvement in mtDNA synthesis regardless of the cell cycle stage. While TK2 expression is cell cycle-independent, the lower expression of TK2 in PSCs, despite their vigorous cell cycle, is likely due to the metabolic shift toward pluripotency maintenance and a preference for nucleic acid synthesis through the pentose phosphate pathway, which is derived from the metabolic transition to aerobic glycolysis.\u003c/p\u003e \u003cp\u003eBased on our findings, we recognized the significance of reduced TK2 expression as a factor that regulates metabolic transitions during the reprogramming process. To gain a deeper understanding of stochastic reprogramming, we sought to identify upstream regulators of TK2. By analyzing publicly available literature, we confirmed that p53 directly binds to the TK2 promoter, thereby exerting its control. The importance of p53 expression and acetylation in the reprogramming and maintenance of pluripotency is well known \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. SIRT1, a member of an extensive family of histone deacetylases, regulates p53 activity, influences the fate of numerous cells, and maintains homeostasis. One reason for the extremely low reprogramming efficiency is that all reprogramming factors are associated with the cell cycle, particularly c-Myc and klf4, which are potent oncogenic proteins, leading to the potential activation of p53 during reprogramming \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Activated p53 halts the cell cycle, induces cell death, and promotes aging, thereby inhibiting cell reprogramming. Consequently, the regulation of p53 activity during reprogramming is a major bottleneck in reprogramming technology. As a regulator capable of controlling p53 activity from an upstream position, SIRT1 plays a key role in maintaining genomic stability and inducing metabolic transitions during the reprogramming process. Specifically, SIRT1 is directly transcribed by OCT4; therefore, ectopically expressed OCT4 increases the expression of SIRT1, reducing the acetylation of p53. It is predicted that the decrease in the number of mtDNA copies and expression of TK2 during somatic cell reprogramming is a consequence of OCT4 activity \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInitially, it was expected that TK2 inhibition would improve the reprogramming efficiency, as it was reported to induce the Warburg effect and mitochondrial function. However, there is a potential for adverse effects on somatic cell reprogramming processes when directly blocking TK2 expression. Tk2 mutant mice (Tk2(KI/KI)) and conditions associated with mtDNA depletion exhibit high AMPK activity [28]. Moreover, AMPK acts as a metabolic barrier, inhibiting somatic cell reprogramming [29]. During somatic cell reprogramming by OSKM, decreased mitochondrial function leads to reduced ATP levels, which may trigger an energy homeostatic response mediated by AMPK. This suggests that energy metabolism and homeostatic mechanisms negatively affect the acquisition of pluripotency during cell reprogramming. Consequently, the notable reduction in TK2 postulates with the utilization of AZT does not significantly contribute to enhancing reprogramming efficiency (results not presented). This suggests that a full reduction in TK2 may induce metabolic stress, DNA damage, and replication stress during the reprogramming process, potentially negatively affecting the acquisition of pluripotency. The primary challenge to investigating alterations in reprogramming efficacy with AZT lies in the possible adverse impacts of directly inhibiting TK2 expression during somatic cell reprogramming. Given the intricacy of cellular mechanisms, the administration of AZT may lead to unforeseen repercussions or off-target effects linked to TK2 suppression, potentially influencing the overarching outcomes. However, iPSCs can also be successfully established in TK2 mutations fibroblasts \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It cannot be ruled out that these results are predominantly the result of cells that completed metabolic adaptation after TK2 knockout. Therefore, direct inhibition of TK2 expression should be carefully fine-tuned in the context of somatic cell reprogramming.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study sheds new light on metabolic transition as a crucial element in the regulation of mtDNA copy numbers during the transition from OXPHOS to aerobic glycolysis, overcoming one of the major barriers to dedifferentiation. Furthermore, we revealed, for the first time, that the SIRT1-p53 axis plays a pivotal role in lowering TK2 expression, impacting metabolic transitions, and the acquisition of pluripotency.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Research Foundation of Korea \u003cb\u003e(NRF-2020R1A6A3A13077021, RS-2023-00220207)\u003c/b\u003e and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government \u003cb\u003e(KFRM-2022-00070557).\u003c/b\u003e This research was supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT \u003cb\u003e(24-BR-02-04).\u003c/b\u003e Chung-Ang University Graduate Research Scholarship (Academic Scholarship for College of Biotechnology and Natural Resources) in 2023.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eWe express our sincere gratitude to Davide Cacciarelli from the Broad Institute for generously providing the hiF-T cell line used in this study for human reprogramming research. We would also like to thank the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute for their assistance with microscopy. Special thanks go to STEMOPIA.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eSupplementary information accompanies the manuscript on the Experimental \u0026amp; Molecular Medicine website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/emm/\u003c/span\u003e\u003cspan address=\"http://www.nature.com/emm/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHanna, J. \u003cem\u003eet al.\u003c/em\u003e Direct Cell Reprogramming Is a Stochastic Process Amenable to Acceleration. Nature. 462, 595\u0026ndash;601 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamanaka, S. Elite and Stochastic Models for Induced Pluripotent Stem Cell Generation. Nature. 460, 49\u0026ndash;52 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi, K. \u0026amp; Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 126, 663\u0026ndash;676 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi, K. \u003cem\u003eet al.\u003c/em\u003e Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 131, 861\u0026ndash;872 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrkin, S. H. \u0026amp; Hochedlinger, K. Chromatin Connections to Pluripotency and Cellular Reprogramming. Cell. 145, 835\u0026ndash;50 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyer, L. A. \u003cem\u003eet al.\u003c/em\u003e Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells. Cell. 122, 947\u0026ndash;56 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshida, T., Nakao, S., Ueyama, T., Harada, Y. \u0026amp; Kawamura, T. Metabolic Remodeling During Somatic Cell Reprogramming to Induced Pluripotent Stem Cells: Involvement of Hypoxia-Inducible Factor 1. Inflamm Regen. 40, 8 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanopoulos, A. D. \u003cem\u003eet al.\u003c/em\u003e The Metabolome of Induced Pluripotent Stem Cells Reveals Metabolic Changes Occurring in Somatic Cell Reprogramming. Cell Res. 22, 168\u0026ndash;77 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarum, S. \u003cem\u003eet al.\u003c/em\u003e Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts. PLoS One. 6, e20914 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrigione, A., Fauler, B., Lurz, R., Lehrach, H. \u0026amp; Adjaye, J. The Senescence-Related Mitochondrial/Oxidative Stress Pathway Is Repressed in Human Induced Pluripotent Stem Cells. \u003cem\u003eStem Cells\u003c/em\u003e. 28, 721\u0026thinsp;\u0026ndash;\u0026thinsp;33 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmes, C. D. \u003cem\u003eet al.\u003c/em\u003e Somatic Oxidative Bioenergetics Transitions into Pluripotency-Dependent Glycolysis to Facilitate Nuclear Reprogramming. \u003cem\u003eCell Metab\u003c/em\u003e. 14, 264\u0026thinsp;\u0026ndash;\u0026thinsp;71 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMathieu, J. \u003cem\u003eet al.\u003c/em\u003e Hypoxia-Inducible Factors Have Distinct and Stage-Specific Roles During Reprogramming of Human Cells to Pluripotency. Cell Stem Cell. 14, 592\u0026ndash;605 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrigione, A. \u003cem\u003eet al.\u003c/em\u003e Hif1α Modulates Cell Fate Reprogramming through Early Glycolytic Shift and Upregulation of Pdk1-3 and Pkm2. \u003cem\u003eStem Cells\u003c/em\u003e. 32, 364\u0026thinsp;\u0026ndash;\u0026thinsp;76 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansson, J. \u003cem\u003eet al.\u003c/em\u003e Highly Coordinated Proteome Dynamics During Reprogramming of Somatic Cells to Pluripotency. Cell Rep. 2, 1579\u0026ndash;92 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVazquez-Martin, A. \u003cem\u003eet al.\u003c/em\u003e Mitochondrial Fusion by Pharmacological Manipulation Impedes Somatic Cell Reprogramming to Pluripotency: New Insight into the Role of Mitophagy in Cell Stemness. Aging (Albany NY). 4, 393\u0026ndash;401 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBukowiecki, R., Adjaye, J. \u0026amp; Prigione, A. Mitochondrial Function in Pluripotent Stem Cells and Cellular Reprogramming. Gerontology. 60, 174\u0026ndash;82 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSon, M. J. \u003cem\u003eet al.\u003c/em\u003e Mitofusins Deficiency Elicits Mitochondrial Metabolic Reprogramming to Pluripotency. Cell Death Differ. 22, 1957\u0026ndash;69 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrieto, J. \u003cem\u003eet al.\u003c/em\u003e Early Erk1/2 Activation Promotes Drp1-Dependent Mitochondrial Fission Necessary for Cell Reprogramming. Nat Commun. 7, 11124 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmes, C. D., Nelson, T. J. \u0026amp; Terzic, A. Energy Metabolism in Nuclear Reprogramming. \u003cem\u003eBiomark Med\u003c/em\u003e. 5, 715\u0026thinsp;\u0026ndash;\u0026thinsp;29 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuhr, S. T. \u003cem\u003eet al.\u003c/em\u003e Mitochondrial Rejuvenation after Induced Pluripotency. PLoS One. 5, e14095 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. M. \u003cem\u003eet al.\u003c/em\u003e Induced Pluripotent Stem Cell Models of Zellweger Spectrum Disorder Show Impaired Peroxisome Assembly and Cell Type-Specific Lipid Abnormalities. Stem Cell Res Ther. 6, 158 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong, L. \u003cem\u003eet al.\u003c/em\u003e Human Induced Pluripotent Stem Cell Lines Show Stress Defense Mechanisms and Mitochondrial Regulation Similar to Those of Human Embryonic Stem Cells. Stem Cells. 28, 661\u0026ndash;673 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelogrudov, G. \u0026amp; Hatefi, Y. Catalytic Sector of Complex I (Nadh:Ubiquinone Oxidoreductase): Subunit Stoichiometry and Substrate-Induced Conformation Changes. Biochemistry. 33, 4571\u0026ndash;6 (1994).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan, E. M. \u003cem\u003eet al.\u003c/em\u003e Live Cell Imaging Distinguishes Bona Fide Human Ips Cells from Partially Reprogrammed Cells. Nature Biotechnology. 27, 1033\u0026ndash;1037 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, X. \u0026amp; St John, J. C. Modulation of Mitochondrial DNA Copy Number in a Model of Glioblastoma Induces Changes to DNA Methylation and Gene Expression of the Nuclear Genome in Tumours. Epigenetics \u0026amp; Chromatin. 11, 53 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartesaghi, S. \u003cem\u003eet al.\u003c/em\u003e Inhibition of Oxidative Metabolism Leads to P53 Genetic Inactivation and Transformation in Neural Stem Cells. Proc Natl Acad Sci U S A. 112, 1059\u0026ndash;64 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCacchiarelli, D. \u003cem\u003eet al.\u003c/em\u003e Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency. Cell. 162, 412\u0026ndash;424 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, K. R. \u003cem\u003eet al.\u003c/em\u003e Chipbase V2.0: Decoding Transcriptional Regulatory Networks of Non-Coding Rnas and Protein-Coding Genes from Chip-Seq Data. Nucleic Acids Res. 45, D43-D50 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebedeva, M. A., Eaton, J. S. \u0026amp; Shadel, G. S. Loss of P53 Causes Mitochondrial DNA Depletion and Altered Mitochondrial Reactive Oxygen Species Homeostasis. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Bioenergetics\u003c/em\u003e. 1787, 328\u0026ndash;334 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadivoyevitch, T. \u003cem\u003eet al.\u003c/em\u003e Dntp Supply Gene Expression Patterns after P53 Loss. Cancers (Basel). 4, 1212\u0026ndash;24 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLonergan, T., Bavister, B. \u0026amp; Brenner, C. Mitochondria in Stem Cells. Mitochondrion. 7, 289\u0026ndash;296 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBitter, E. E., Townsend, M. H., Erickson, R., Allen, C. \u0026amp; O\u0026rsquo;Neill, K. L. Thymidine Kinase 1 through the Ages: A Comprehensive Review. Cell \u0026amp; Bioscience. 10, 138 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Z. N., Chung, S. K., Xu, Z. \u0026amp; Xu, Y. Oct4 Maintains the Pluripotency of Human Embryonic Stem Cells by Inactivating P53 through Sirt1-Mediated Deacetylation. Stem Cells. 32, 157\u0026ndash;65 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, Y. L. \u003cem\u003eet al.\u003c/em\u003e Sirtuin 1 Facilitates Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts through the Mir-34a and P53 Pathways. PLoS One. 7, e45633 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, A. K. \u003cem\u003eet al.\u003c/em\u003e P53 Regulates Cell Cycle and Micrornas to Promote Differentiation of Human Embryonic Stem Cells. PLoS Biol. 10, e1001268 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, T. \u003cem\u003eet al.\u003c/em\u003e P53 Induces Differentiation of Mouse Embryonic Stem Cells by Suppressing Nanog Expression. Nat Cell Biol. 7, 165\u0026ndash;71 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, X., Wu, S., Li, B., Xu, Y. \u0026amp; Liu, J. Functions of P53 in Pluripotent Stem Cells. Protein Cell. 11, 71\u0026ndash;78 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHernandez-Ainsa, C. \u003cem\u003eet al.\u003c/em\u003e Generation of an Induced Pluripotent Stem Cell Line from a Compound Heterozygous Patient in Tk2 Gene. Stem Cell Res. 59, 102632 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5148938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5148938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReprogramming somatic cells into human induced pluripotent stem cells entails profound intracellular changes, including modifications in mitochondrial metabolism and a decrease in the mitochondrial DNA copy number. However, the mechanisms underlying this decline in mitochondrial DNA copy number during reprogramming remain unclear. In this study, we aimed to elucidate these underlying mechanisms. Through meta-analysis of numerous RNA sequencing datasets, we identified the genes responsible for the reduction in mitochondrial DNA. We investigated the functions of these identified genes and examined their regulatory mechanisms. Particularly, the thymidine kinase 2 (\u003cem\u003eTK2\u003c/em\u003e) gene, required for mitochondrial DNA synthesis and found in the mitochondria, exhibits diminished expression in human pluripotent stem cells compared with that in somatic cells. TK2 was substantially downregulated during reprogramming and markedly upregulated during differentiation. Collectively, the reduction in TK2 levels influences a decrease in mitochondrial DNA copy number and participates in shaping the metabolic characteristics of human pluripotent stem cells. However, contrary to our expectations, treatment with a TK2 inhibitor impaired somatic cell reprogramming. These results suggest that reduced TK2 expression may result from metabolic conversion during somatic cell reprogramming.\u003c/p\u003e","manuscriptTitle":"Changes in mitochondrial thymidine metabolism and mtDNA copy number during induced pluripotency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-07 05:30:26","doi":"10.21203/rs.3.rs-5148938/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-11-29T07:04:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-23T18:12:00+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-23T17:12:00+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-15T07:42:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-14T04:02:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-01T02:00:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-10-29T07:32:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-10-29T01:30:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-10T07:20:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2024-10-10T07:10:15+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-09-26T02:23:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-25T04:59:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"021dc277-9be5-4190-9f9a-66f92625fb02","owner":[],"postedDate":"November 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39533086,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":39533087,"name":"Biological sciences/Stem cells/Reprogramming"}],"tags":[],"updatedAt":"2025-06-27T07:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-5148938","link":"https://doi.org/10.1038/s12276-025-01476-3","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-06-26 04:00:00","publishedOnDateReadable":"June 26th, 2025"},"versionCreatedAt":"2024-11-07 05:30:26","video":"","vorDoi":"10.1038/s12276-025-01476-3","vorDoiUrl":"https://doi.org/10.1038/s12276-025-01476-3","workflowStages":[]},"version":"v1","identity":"rs-5148938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5148938","identity":"rs-5148938","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}