Author contributions: A.P.: conception and design, data collection, analysis and interpretation, manuscript writing; B.L., H.K., E.A.S., and M.W.: data collection; H.L.: infrastructure support; M.R.: data interpretation; B.T.: data collection and interpretation; J.A.: data interpretation, manuscript editing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS July 5, 2011.
Human induced pluripotent stem cells (iPSCs) have been recently found to harbor genomic alterations. However, the integrity of mitochondrial DNA (mtDNA) within reprogrammed cells has yet to be investigated. mtDNA mutations occur at a high rate and contribute to the pathology of a number of human disorders. Furthermore, the lack of mtDNA integrity may alter cellular bioenergetics and limit efficient differentiation. We demonstrated previously that the derivation of iPSCs is associated with mitochondrial remodeling and a metabolic switch towards glycolysis. Here, we have discovered that alterations of mtDNA can occur upon the induction of pluripotency. Massively parallel pyrosequencing of mtDNA revealed that human iPSCs derived from young healthy donors harbored single base mtDNA mutations (substitutions, insertions, and deletions), both homoplasmic (in all mtDNA molecules) and heteroplasmic (in a fraction of mtDNAs), not present in the parental cells. mtDNA modifications were mostly common variants and not disease related. Moreover, iPSC lines bearing different mtDNA mutational loads maintained a consistent human embryonic stem cell–like reprogramming of energy metabolism. This involved the upregulation of glycolytic enzymes, increased glucose-6-phosphate levels, and the over-expression of pyruvate dehydrogenase kinase 1 protein, which reroutes the bioenergetic flux toward glycolysis. Hence, mtDNA mutations within iPSCs may not necessarily impair the correct establishment of pluripotency and the associated metabolic reprogramming. Nonetheless, the occurrence of pathogenic mtDNA modifications might be an important aspect to monitor when characterizing iPSC lines. Finally, we speculate that this random rearrangement of mtDNA molecules might prove beneficial for the derivation of mutation-free iPSCs from patients with mtDNA disorders. STEM CELLS 2011; 29:1338–1348
Defects in mitochondrial DNA (mtDNA) are a frequent cause of genetic disease, as they are estimated to develop in 1 in 5,000 individuals . Unfortunately, due to the paucity of animal and cellular models, the genotype-phenotype correlation of mitochondrial disorders is not yet entirely elucidated . Induced pluripotent stem cells (iPSCs)  derived from individuals with mtDNA disorders might then represent an interesting modeling tool, as they would allow to investigate the interplay between mitochondrial and nuclear genomes and the effects of particular mutations on distinct cellular identities. However, before applying iPSC technology in the context of mtDNA diseases, it is essential to determine the effects of the reprogramming process on mitochondrial genome integrity of healthy somatic cells.
A plethora of recent findings revealed the presence of reprogramming-associated genomic alterations [4–8], suggesting that the derivation of iPSCs may carry the risk of inducing mutations. This is particularly relevant for mtDNA, because it is considered to be highly susceptible to mutagenic events due to the lack of protective histones, the poor fidelity of the DNA polymerase gamma, and the proximity to free radical production , which has been found increased during the early steps of reprogramming .
Mitochondrial genetics is fundamentally different from Mendelian genetics. mtDNA is transmitted along the maternal lineage and its genes are not transcribed individually but rather as polycistronic RNA precursors . Importantly, the mitochondrial genome exhibits a polyploid nature, that is, it exists in several copies within every single cell. When all copies of mtDNA are identical, the state is defined as homoplasmy, while heteroplasmy describes the situation in which there is a mixture of two mtDNA genotypes. The heteroplasmic level is of critical significance, as the pathogenic phenotypes are believed to occur only when the number of mutated molecules reaches a certain threshold . Finally, mtDNA variants can be unequally distributed during proliferative division. This effect, known as replicative segregation, characterizes the mitochondrial genetic bottleneck, a phenomenon in which mothers harboring a mixture of mutated and wild-type mtDNAs transmit varying proportions of mutated and wild-type mtDNA to their offspring [13, 14].
The mitochondrial genome encodes 13 proteins contributing to complex I, III, IV, and V of the electron transport chain (ETC), which generates ATP through oxidative phosphorylation (OXPHOS) . Hence, mtDNA integrity plays a relevant role for cellular bioenergetics. Large-scale deletions are in fact associated with reduced ATP generation [16, 17] and mtDNA diseases often affect tissues with high energy requirements, such as the nervous system and skeletal muscle .
We demonstrated previously that human somatic mitochondria undergo a complex remodeling within iPSCs, as they adapt their morphology and functionality to acquire the features of mitochondria within human embryonic stem cells (hESCs) . These mitochondrial changes also impacted the cellular bioenergetic profile, which shifted from OXPHOS to glycolysis upon reprogramming and returned to OXPHOS during subsequent differentiation [18, 19]. Other groups confirmed these results and showed that mitochondria within human iPSCs exhibits low oxidative stress  and energetic rejuvenation . Moreover, small molecules inducing metabolic switch toward glycolytic metabolism enhanced reprogramming efficiency .
In this work, we sought to investigate mtDNA integrity in healthy human reprogrammed cells. As the polyploid nature of mitochondrial genome poses a challenge for conventional sequencing, we used next-generation sequencing (NGS) technology, which enables the discovery of heteroplasmy at high resolution [23–25]. We detected several homoplasmic and heteroplasmic mtDNA mutations within distinct iPSC lines when compared with their parental fibroblast cells. Importantly, the level of heteroplasmy varied between distinct iPSC lines obtained from the same parental fibroblasts, suggesting that reprogramming might induce replicative segregation of mitochondria in a fashion resembling the mitochondrial genetic bottleneck of embryonic development. This phenomenon could potentially be exploited for the derivation of mutation-free iPSC lines from patients affected by mtDNA disorders. Finally, by combining transcriptional, metabolic, and functional bioenergetic data, we determined that the detected mtDNA variability among our iPSC lines did not affect the reprogramming-associated reconfiguration of energy metabolism. iPSC lines harboring different mtDNA mutations exhibited similar hESC-like bioenergetic profiles and, when compared with the parental fibroblasts, showed equivalent hESC-like elevation of glucose-6-phosphate (G6P), the largest hub of carbon metabolism , and over-expression of pyruvate dehydrogenase kinase (PDK1), which inhibits the entry of pyruvate into the mitochondria, thereby rerouting the energetic flux toward glycolysis .
Overall, the findings, although conducted on a limited number of human iPSC lines, indicate that cellular reprogramming might be associated with modifications within the mitochondrial genome and thus suggest an additional aspect to monitor during the production of clinical-grade iPSCs.
MATERIALS AND METHODS
Culture Conditions and Derivation of iPSCs
hESC lines H1 and H9 (WiCell, www.wicell.org) and iPSCs were cultured as described previously . Neonatal foreskin fibroblasts BJ and HFF1 were bought from ATCC (www.atcc.org; #CRL-2522 and # SCRC-1041, respectively). Adult NFH2 fibroblasts were obtained from a healthy 84-year-old woman, after approval of the study protocol by the ethic committee and written informed consent, at the University Hospital of Dessau (Prigione A, Hossini AM et al. unpublished observation). All cultures were kept in a humidified atmosphere of 5% CO2 at 37°C under atmospheric oxygen condition.
HFF1-iPSCs (lines iPS2 and iPS4) were previously generated using the Yamanaka retroviral cocktail . BJ-iPSCs (lines iB4 and iB5) were obtained using the same approach. Induced pluripotency in iB4 and iB5 was confirmed by using embryoid body-based in vitro differentiation and the in vivo teratoma assay. Teratoma experiments were carried out by EPO-Berlin Gmbh and histological analysis was performed by a certified pathologist. Fingerprinting analysis was assayed to verify somatic origin of iPSCs using the primer sets D10S1214 and D21S2055 . For detection of possible karyotype abnormalities in iPSCs, chromosomal analysis after GTG-banding was executed at the Human Genetic Center of Berlin. Derived fibroblasts (DFs) were obtained from hESCs and iPSCs as indicated previously .
NGS of mtDNA
Generation of an Amplicon Library
Total DNA was isolated with FlexiGene DNA kit (Qiagen, Hilden, Germany, www.qiagen.com) from BJ (passage 4), HFF1 (passage 4), BJ-derived iB4 and iB5 (both passage 18), and HFF1-derived iPS2 and iPS4 (both passage 18). DNA (50 ng) was used as polymerase chain reaction (PCR) template. PCR reactions contained 1× Phusion HF buffer, 3% dimethyl sulfoxide (DMSO), and 0.6 Units of proof-reading Phusion Hot Start II High Fidelity Polymerase (all from Finnzymes, Espoo, Finland, www.finnzymes.com). The reactions were carried out using Dyad Thermal Cycler (BioRad, Hercules, CA, www.bio-rad.com), according to the following program: 98°C for 30 seconds, followed by 30 cycles of 98°C for 10 seconds/58°C for 30 seconds/72°C for 30 seconds, and a final extension step at 72°C for 5 minutes. Primers sequences are reported in Supporting Information Table 1.
454 Sequencing and Data Analysis
PCR fragments were purified, pooled, and universal sequencing adaptors ligated. After emulsion and template annealing, beads were incubated with Bst DNA polymerase, apyrase, and single-stranded binding proteins. Pyrosequencing was performed on a 70 × 75 mm picotiter plate using a gasket with distinct compartments for physical separation of the samples. The picotiter plate was inserted in the flow cell and subjected to pyrosequencing on the Genome Sequencer FLX 454 instrument (Roche, Branford, CT, www.roche.com), as described previously . For each deoxynucleotide triphosphate flow, a single image was captured by a charge-coupled device camera on the sequencer. The images were first analyzed to identify DNA bead-containing wells and to compute associated signal intensities and then processed for chemical and optical crosstalk, phase errors, and read quality, before base calling was carried out for each template bead. After default raw data processing, a resequencing trimming filter was applied to increase the data output (parameters doValleyFilterTrimBack = false, vfBadFlowThreshold = 6, vfLastFlowToTest = 168, errorQscoreWindowTrim = 0.01).
Mapping 454 Reads to Reference Genome and Detection of Variants
Approximately 0.8 million sequences were generated for a total of 662 million bases. The revised Cambridge Reference Sequence (rCRS) (GenBank accession NC_012920) was used as reference for alignment and detection of variants was performed using the GS Reference Mapper Version 2.3 (Roche). Only the high confidence differences (HCDiff) of the GS Mapper software were used as basis for variant detection. HCDiff callings presumed at least three reads with the variant with both forward and reverse reads. Alternatively, the quality scores at variable positions must be more than 20 (or more than 30, if a homopolymer of five or more bases was involved). As additional quality criteria, we used only variants with a coverage of more than 10× of high-quality reads. With respect to rCRS, a variant was defined heteroplasmic when its frequency was ≥10% and homoplasmic if its frequency was ≥90% . The following databases were used to identify mtDNA mutations and mtDNA haplogroups: mitomap (http://www.mitomap.org), mtDB (http://www.genpat.uu.se/mtDB/), and phyotree (http://www.phylotree.org/).
Global Gene Expression Analysis
Total RNA was quality-checked by Nanodrop analysis (Nanodrop Technologies, Wilmington, DE, www.nanodrop.com) and a quantity of 500 ng was used as input. Illumina BeadStation 500 platform (Illumina, San Diego, CA, www.illumina.com) was used for hybridizations, washing, Cy3-streptavidin staining, and scanning. Complementary ribonucleic acids derived from the following samples were hybridized in duplicate onto Illumina human-8 BeadChips version 3: BJ, HFF1, H1, H9, iB4, iB5, PS2, and iPS4. Data analysis was carried out using the BeadStudio software 3.0. Genes were considered significantly expressed with detection p values ≤.01, differential p values ≤.01 (Illumina custom method), and fold change ratio >1.5. Heatmaps were generated using Microarray Software Suite TM4 (www.tm4.org). The list of genes involved in cellular bioenergetics was derived from SA Biosciences PCR arrays (Human Glucose Metabolism PCR Array, www.sabiosciences.com). Gene expression results have been deposited in the Gene Expression Omnibus database (record GSE26575).
Liquid Chromatography Tandem Mass Spectrometry
Intermediates of glycolysis and the pentose phosphate pathway (PPP) were quantified by liquid chromatography tandem mass spectrometry LC-MS/MS as described earlier . In brief, metabolites were extracted in Hanks' balanced salt solution containing 2% perchloric acid and spiked with an isotope-labeled internal standard (13C6-glucose-6P). Proteins were precipitated after neutralization with a phosphate buffer. From the clear supernatant, 10 μL was injected onto the LC column. Intermediates of the PPP were separated on a C18 column using a linear water/acetonitrile gradient containing octylammonium acetate (500 mg/l, pH 7.5) as ion pairing reagent, with a flow rate of 1 ml/minute. Metabolite detection was performed using an API-3000 tandem mass spectrometer (AB Sciex, Nieuwerkerk aan den Ijssel, The Netherlands, www.absciex.com), equipped with a turbo ion electrospray source operating in the negative mode. Data were obtained by operating the tandem mass spectrometer in the multiple reaction monitoring mode using (Q1/Q3) transitions as indicated previously .
Quantitative Real-Time PCR
Real-Time PCR was performed in 384- or 96-Well Optical Reaction Plates (Applied Biosystems, Foster City, CA, www.appliedbiosystems.com) using SYBR Green PCR Master Mix (Applied Biosystems). Reactions were carried out on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Triplicate amplifications were carried out for each target gene with three wells serving as negative controls. Quantification was carried out using the comparative Ct method (ABI instruction manual), normalized over glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and presented as a log2 values with respect to the biological controls. Human Embryonic Stem Cells StellARray qPCR array (Lonza, Cologne, Germany, www.lonza.com) was used to confirm the expression of key pluripotency-associated genes in BJ-iPSC lines. Values of fold change ratio over somatic fibroblasts were obtained by GPR analysis. mtDNA copy number was quantified using ND5 and CF gene targets for mitochondrial and nuclear genome, respectively, as described previously . The list of all primers used for real-time PCR experiments is reported in Supporting Information Table 2.
Live quantification of cellular and mitochondrial bioenergetics was performed using Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, www.seahorsebio.com), as described previously . Preliminary experiments were carried out to prove that hESCs and iPSCs maintained the expression of key pluripotency-associated proteins when grown on Matrigel-coated XF96 well plates (data not shown). Assays were initiated by removing the growth medium and replacing it with unbuffered media. The cells were incubated at 37°C for 30 minutes to allow media temperature and pH to reach equilibrium before starting the baseline measurement. After having obtained the basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), the cells were metabolically perturbed by the additions of three different compounds in succession: oligomycin (1 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (0.3 μM), and rotenone (1 μM) (all from Sigma, St. Louis, MO, www.sigmaaldrich.com). At the end of each assay, cells were trypsinized and the number of viable cells was counted.
Cellular ATP content was determined using the ATPLite bioluminescence luciferase-based assay (Perkin Elmer, Waltham, MA, www.perkinelmer.com), as described previously . Briefly, 100,000 cells were used as input and luminescence was quantified with a luminometer (Berthold Technologies, Bad Wildbad, Germany, www.berthold.com), following the manufacturer's manual. Every sample was measured in triplicate and the results are presented as nanomoles of ATP per cell.
Immunofluorescence and Alkaline Phosphatase Staining
Immunocytochemistry and alkaline phosphatase (#SCR004, Millipore, Schwalbach, Germany, www.millipore.com) staining were performed as previously indicated . Primary antibodies included stage-specific embryonic antigen 1 (SSEA1) and SSEA4, TRA-1-60, and TRA-1-81 from the hESC characterization tool (all 1:100, Millipore #SCR004), NANOG (1:100, #ab62734, Abcam, Cambridge, UK, www.abcam.com), smooth muscle actin (1:100, #M0851, Dako, Glostrup, Denmark, www.dako.com), alpha feto protein (1:100, Sigma #WH0000174M1), SOX17 (1:50, #AF1924, R&D Systems, Minneapolis, MN, www.rndsystems.com), PAX6 (1:300, #PRB-278P, Covance, Princeton, NJ, www.covance.com), NESTIN (1:200, Millipore #MAB5326), and TUJ-1 (1:1,000, Sigma #T8660). Secondary antibodies were conjugated with either Alexa 488 or Alexa 594 (#A11001, A11055, A21201, A21468, A11005, A21442, Invitrogen, Darmstadt, Germany, www.invitrogen.com). Coverslips were mounted using Dako fluorescent mounting medium (Dako #S3023) and visualized using a confocal microscope LSM 510 (Zeiss, Jena, Germany, www.zeiss.de).
Western Blotting Analysis
Total cell protein extracts were obtained using a modified radio immunoprecipitation assay buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% IGEPAL) supplemented with complete protease inhibitor cocktail (Roche) added just before use. Protein concentration was determined according to the Bradford method. Equal quantities of proteins were separated by electrophoresis in a 10% SDS-polyacrylamide gel (BioRad). Primary antibodies anti-PDK1 (1:1,000, #KAP-PK112, Enzo Life Sciences, Lörrach, Germany, www.enzolifesciences.com) and GAPDH (1:5,000, #4300, Ambion, Austin, TX, USA, www.ambion.com) were used with the suitable horseradish peroxidase–conjugated secondary antibodies. Bound antibodies on nitrocellulose membrane (GE Healthcare, Munich, Germany, www.gehealthcare.com) were detected using the enhanced chemiluminescent substrate (GE Healthcare) and membranes were stripped using Restore Western Blot (Thermo Scientific, Rockford, IL, www.thermo.com).
NGS of mtDNA in Healthy Human iPSCs
Two sets of healthy human iPSCs, derived from the neonatal foreskin fibroblasts HFF1 and BJ, were used in this study. HFF1-iPSCs (lines iPS2 and iPS4) were previously generated via retroviral transduction of the Yamanaka cocktail of factors . BJ-iPSCs (lines iB4 and iB5) were obtained in a similar fashion and underwent full characterization. BJ-iPSCs expressed hESC-specific protein markers (Fig. 1A) were karyotypically normal (Supporting Information Fig. 1A), exhibited the same genetic fingerprinting pattern of BJ fibroblasts (Supporting Information Fig. 1B), and efficiently differentiated into the three germ layers both in vitro (Supporting Information Fig. 1C) and in vivo (Supporting Information Fig. 1D). BJ-iPSCs also acquired a transcriptional signature similar to that of the hESC lines H1 and H9 (Pearson correlation value: 0.8309) and distinct from that of fibroblasts (Pearson correlation value: 0.6660) (Supporting Information Fig. 2A), and expressed key pluripotency-associated genes (Supporting Information Fig. 2B, 2C). Finally, before analyzing the mtDNA sequence of all iPSC lines and corresponding fibroblasts, we confirmed that BJ-iPSCs, like HFF1-iPSCs, displayed a decreased mtDNA copy number when compared with the parental cells, in agreement with previous findings [18, 20] (Fig. 1B).
mtDNA sequencing was carried out using Roche 454 sequencing, which showed similar reliability coupled with higher sensitivity for the discovery of low level heteroplasmies in comparison with Sanger sequencing . An amplicon library was first created using two sets of 25 primer pairs (primer sets A and B, Supporting Information Table 1), each set designed to encompass the entire mitochondrial genome . All primers successfully generated single amplicons of approximately 650 base pair (Supporting Information Fig. 3). PCR fragments were then purified, pooled, ligated with universal sequencing adaptors, and subjected to massively parallel pyrosequencing (Fig. 1C). The 454 sequencing output had an average read length of 405 bases per read and an average sequencing content of 96.5 megabases per mtDNA genome. More than 99.6% of all reads mapped to the mitochondrial reference sequence (rCRS) with a total average coverage per base of 5,937 X. This led to a single continuous sequence contig of 16,560 bases per sample, thus reaching complete covering of the mitochondrial target region (Supporting Information Table 3). After having determined all mtDNA variants in the individual samples using the rCRS as a reference, we compared each iPSC line with the respective fibroblasts (BJ for iB4 and iB5 and HFF1 for iPS2 and iPS4) to identify mtDNA changes occurring upon reprogramming.
Homoplasmic and Heteroplasmic mtDNA Mutations in Human iPSCs
The mitochondrial genome of the four iPSC lines did not exhibit signs of large-scale rearrangement, such as long insertions or long deletions. However, several point mutations were detected within iPSCs when compared with their parental cells (Table 1). Interestingly, the two sets of iPSCs showed a different pattern of mutations, and we could not identify any variant consistently present in all reprogrammed cells and not in fibroblasts (Supporting Information Tables 4, 5). The number of mtDNA mutations also differed between the two sets, being approximately 20 in BJ-iPSCs and approximately 80 in HFF1-iPSCs (Table 1). The reason for this variability is not clear and could be due to the features of the cell type of origin or to reprogramming-related factors, as the two sets of iPSCs were derived at different times with different retroviral batches. Future studies are warranted to determine whether this mtDNA sequence variability is a common aspect of all iPSCs or it is influenced by differences in the cellular source or in the reprogramming strategy used.
Table 1. Summary of mitochondrial DNA mutations identified within human induced pluripotent stem cells
The various mtDNA mutations were classified according to their frequency in homoplasmic and heteroplasmic mutations (Table 1). Homoplasmic variants included single base substitutions, single base insertions, and single base deletions (Supporting Information Table 4). Base substitutions were highly frequent within HFF1-iPSCs, while the other types of homoplasmic or heteroplasmic variants occurred at approximately equal numbers in all iPSC lines (Table 1). In some cases, such as position 750 for the BJ set, fibroblasts contained mtDNA variants with respect to the rCRS and, upon reprogramming, these variants could be either maintained or lost in different iPSC lines (Fig. 2A; Supporting Information Table 4). In other cases, such as position 6,776 for the HFF1 set, iPSCs acquired homoplasmic mtDNA mutations not present in the parental somatic cells (Fig. 2B; Supporting Information Table 4). Interestingly, base substitutions also affected nucleotides critical for the assignment to specific mitochondrial haplogroups . For example, in BJ fibroblasts, whose haplogroup was calculated to be K1c1, the nucleotide in the haplogroup-defining position 3,480 was G, while it became A in iB4 and was deleted in iB5 (Supporting Information Table 4).
The level of heteroplasmy of heteroplasmic variants within fibroblasts remained unaltered (±5%) in the reprogrammed cells only in a small percentage of cases, while most frequently it sustained a consistent change (Table 1; Supporting Information Table 5). For example, at nucleotide position 9,903, BJ fibroblasts showed a heteroplasmic variant whose level of heteroplasmy was 45%. This level increased to 100% in iB4 and decreased to 0% in iB5 (Fig. 2C; Supporting Information Table 5). In addition, iPSCs exhibited heteroplasmic mutations that were not contained in the original fibroblast samples. At the nucleotide at position 16,126, for example, HFF1 fibroblasts were homoplasmic for T, while HFF1-iPSCs acquired heteroplasmic T>C mutations (48% C and 50% C in iPS2 and iPS4, respectively) (Supporting Information Table 5).
mtDNA mutations within iPSCs affected both noncoding genes, including components of the control region or of the translational machinery, such as transfer ribonucleic acids, ribosomal ribonucleic acids (12s and 16s), and genes encoding for proteins contributing to the ETC (Table 1; Supporting Information Tables 4, 5). In all iPSC lines, most coding mutations were synonymous, thus not functionally relevant. The nonsynonymous mutations, which gave rise to an amino acid change, affected genes encoding for proteins part of all the four mitochondrial-encoded ETC complexes, with no particular predisposition (Table 1). The nonsynonymous/synonymous ratio was 2 for iB4, 3 for iB5, and 1 for iPS2 and iPS4 (Table 1; Supporting Information Tables 4, 5).
The majority of mtDNA variants within our reprogrammed cells are considered to be “common variants” in the general populations. Of these, only a very small number appeared to be associated with diseases or cancer, based on literature (Table 1; Supporting Information Tables 4, 5). On the other hand, some mutations, mostly heteroplasmies, could not be found in any of the mtDNA databases listing all possible mtDNA sequence variants. Future studies will have to address the possible relevance of these unknown mutations in cellular reprogramming.
mtDNA Sequence Variability Does Not Affect the hESC-like Reprogramming of Energy Metabolism
We previously demonstrated that the process of deriving iPSC induces mitochondria remodeling and metabolic reconfiguration from OXPHOS to glycolysis . In view of this, we aimed to determine whether the differences in the mtDNA profile among iPSC lines could affect the reprogramming of energy metabolism.
At the transcriptional level, we detected a similar upregulation of genes regulating the first steps of the glycolytic cascade in all iPSCs and hESCs in comparison with fibroblasts. These included SLC2A3, encoding GLUT3, which is responsible for the uptake of glucose into the cell (Figs. 3, 4A; Supporting Information Fig. 4), GCK and HK3, whose products are enzymes phosphorylating glucose to G6P (Fig. 3; Supporting Information Fig. 4). Genes of the last steps of glycolysis, such as PGAM2, ENO, PKLR, and LDH were also mainly upregulated in both hESCs and all iPSCs (Fig. 3; Supporting Information Fig. 4). On the other hand, the expression of G6P downstream glycolytic enzymes, including GPI (Figs. 3, 4A; Supporting Information Fig. 4), PFK, and ALDO (Fig. 3; Supporting Information Fig. 4) was reduced in pluripotent stem cells when compared with fibroblasts. These results might imply that in hESCs and iPSCs, regardless of the presence of mtDNA mutations, there might be a parallel induction of the first and last steps of glucose metabolism and a possible accumulation of G6P, which may be diverted into the PPP .
LC-MS/MS was used to simultaneously analyze the intracellular sugar phosphate intermediates of the glycolytic cascade and PPP. G6P, the largest hub of carbon metabolism, was confirmed to be significantly increased in all hESCs and iPSCs when compared with fibroblasts (Fig. 4B). In addition, dihydroxyacetone phosphate was reduced in all pluripotent stem cells, possibly implying an increased flux toward pyruvate generation (Fig. 4B).
Most of the genes encoding for enzymes of the gluconeogenesis pathway were upregulated in all pluripotent stem cells, including MDH1B, FBP1, G6PC (Fig. 3; Supporting Information Fig. 4), and PCK1 (Figs. 3, 4A; Supporting Information Fig. 4). Conversely, genes of the mitochondrial tricarboxylic acid (TCA) cycle, such as ACO, IDH2, SUCLG2, SDH (A and D) (Fig. 3; Supporting Information Fig. 4), and FH (Figs. 3, 4A; Supporting Information Fig. 4) were mainly downregulated in hESCs and iPSCs in comparison with fibroblasts.
Among the regulators of the bioenergetic metabolism, we observed that all pluripotent stem cells showed downregulation of PRKAA1 (Figs. 3, 4A; Supporting Information Fig. 4), which encodes for the catalytic subunit of AMP-activated protein kinase (AMPK) and has been found induced in response to decreased ATP content in mitochondrial-defective cells . It is tempting to speculate that pluripotent stem cells might suppress the activation of AMPK, a metabolic checkpoint switching-off biosynthetic pathways, to avoid anabolic inhibition, similarly to cancer cells [26, 34].
A second metabolic regulator, PDK (PDK1, 3, and 4), was upregulated in hESCs and in all iPSCs despite mtDNA sequence variants (Fig. 3; Supporting Information Fig. 4). PDK1 is of particular interest because it inhibits pyruvate dehydrogenase, thus limiting the pyruvate entry into the TCA cycle . Western blot analysis confirmed increased PDK1 protein expression in hESCs and iPSCs when compared with neonatal fibroblasts (HFF1 and BJ) and with adult fibroblasts (NFH2) (Fig. 4C). Moreover, PDK1 protein levels decreased during subsequent differentiation into fibroblast-like cells (DFs) obtained from hESCs (H1 line), from iPSCs with a low number of mtDNA mutations (iB5 line), and from iPSCs with a high number of mtDNA variants (iPS2 line) (Fig. 4C). Taken together, regardless of variations within mtDNA sequence, all iPSCs acquired a hESC-like transcriptional and metabolic signature indicative of a metabolic transition from respirative to glycolytic metabolism.
Similar hESC-like Bioenergetic Profiles in iPSCs Harboring Different mtDNA Mutations
Finally, we asked whether iPSC lines with comparable metabolic reprogramming could be bioenergetically similar despite differences in mtDNA mutational load. The level of total cellular ATP in all iPSCs was equal to that of hESCs and consensually reduced when compared with fibroblasts (Fig. 5A), in agreement with previous findings . Simultaneous quantification of mitochondrial respiration (OCR) and glycolysis (ECAR) revealed that hESCs and iPSCs with either low (iB5) or high (iPS2) mitochondrial mutational load (Table 1) exhibited an equivalent glycolytic switch, as indicated by the reduction of the OCR/ECAR ratio in comparison with fibroblast cells (Fig. 5B).
Metabolic perturbation with subsequent application of different mitochondrial inhibitors was carried out to shift the energy profile and obtain estimates of different parameters of mitochondrial bioenergetics (Supporting Information Fig. 5). The ATP turnover, the maximal respiration rate, the magnitude of the proton leak, the amount of nonmitochondrial oxygen consumption, and the level of ATP reserve were all equivalent among iPSC lines bearing different mtDNA mutation levels (Fig. 5B). Interestingly, we observed that the acidification rate increased in fibroblasts but not in hESCs and iPSCs, following the exposure to the respiration-blocker oligomycin. Hence, pluripotent stem cells might fully use their glycolytic resources and may not be able to further increase glycolysis upon the blockage of mitochondrial activity (Supporting Information Fig. 3).
Overall, the bioenergetic profile of iPSCs appeared highly similar to the one of hESCs and drastically distinct from their parental somatic cells (Fig. 5B). Nonetheless, the level of ATP reserve in iPSCs was comparable with the one of fibroblasts and much lower than the one exhibited by hESCs (Fig. 5B), potentially suggesting that hESCs and iPSCs might exhibit a different ability in responding to increased energy demands.
Genetic defects of the human mitochondrial genome form the basis of classical mitochondrial syndromes . In addition, growing evidence demonstrates their involvement in the pathology of several other conditions, including diabetes, aging, and neurodegeneration [2, 35, 36]. mtDNA mutations have also been associated with oncogenic transformation  and malignant progression , although their overall role in cancer remains controversial [39, 40]. In this work, we have investigated whether cellular reprogramming might induce mitochondrial mutagenesis in healthy human somatic cells.
We found that distinct iPSC lines harbored various mtDNA point mutations not present in parental fibroblasts. The ratio of nonsynonymous versus synonymous mutations was always equal to one or higher, possibly implying positive selection. Most iPSC-specific mutations were listed as common variants in the general population. Cancer-associated mutations showed similar features and have been suggested to represent mild mtDNA alterations helping the cells to adapt to a new environment, as they have been adaptive during population migration . Accordingly, we found that variations in iPSCs could also occur at haplogroup-defining nucleotide positions.
The heteroplasmy level varied among iPSC lines derived from the same parental cells. Hence, during the cellular replication events accompanying reprogramming, a mitochondrial segregation might occur and the derived cells could harbor a reduced number of mtDNA molecules with variable ratio between wild-type and mutant (Fig. 6). This bottleneck effect might potentially allow the derivation of mtDNA mutation-free iPSC lines from individuals affected by mtDNA disorders. However, to achieve this, it might be necessary to screen a vast number of cell lines and confirm that new mutations are not introduced upon long-term culturing.
Genome-wide sequencing of protein coding regions recently revealed the presence of various point mutations in human iPSCs . Given that mitochondrial genome undergo higher mutation rates when compared with chromosomal DNA , it is perhaps not surprising that we have detected mtDNA mutations within different human iPSC lines. At the same time, it is necessary to investigate the functional significance of these mutations. Our data, combining transcriptional, metabolic, and functional data, revealed that the mtDNA mutations within our iPSCs did not affect their bioenergetic properties. Nonetheless, given the random drift of mitochondria and the consequent variance in the substitution profile, it is likely that other iPSC lines might harbor different alterations of the mitochondrial genome. This is relevant because pathogenic mtDNA mutations and lack of mtDNA integrity can affect cellular energy capacities [16, 17] and compromise efficient stem cell differentiation [42, 43].
By monitoring the energetic consequences of mtDNA sequence variability in iPSCs, we gained new insights into the reprogramming-associated reconfiguration of the metabolic state (Fig. 6). First, G6P was increased in hESCs and iPSCs in comparison with fibroblasts, which might suggest that pluripotent stem cells divert their energy flux from the glycolytic path into the PPP. This may have significant advantages because the PPP not only provides nucleotide precursors essential for anabolic growth but also maintains cellular antioxidant defenses [33, 44]. Furthermore, the protein expression level of PDK1, which promotes glycolytic metabolism by inhibiting the pyruvate entry in the TCA cycle , was consensually elevated in undifferentiated hESCs and iPSCs. In accordance, recent data showed that small molecules activating PDK1 significantly enhanced cellular reprogramming .
Mitochondria within all pluripotent stem cells possessed high coupling efficiency, implying that, although reduced in number and less used for cellular energetics, they are still capable of competent energy generation. This is in agreement with recent findings showing that mitochondrial OXPHOS is required for the proliferation of self-renewing hESCs [45, 46]. On the other hand, iPSCs showed reduced levels of ATP reserve in comparison with hESCs. Thus, iPSCs might not acquire the same ability of hESCs to respond to increased energy demands, which could occur during differentiation. Indeed, differentiated cells from iPSCs exhibited high variability and premature senescence [47, 48]. Our data, demonstrating heterogenic mtDNA rearrangement coupled with a lack of acquisition of high ATP reserve within iPSCs, may help to explain this variability. Perhaps reprogrammed cells might retain not only an epigenetic [49, 50] but also a bioenergetic memory of their somatic origin. Accordingly, somatic cell nuclear transfer, which does not involve somatic mitochondria and does not require bioenergetic reprogramming, showed low heterogeneity and a more faithful establishment of pluripotency [49, 51].
Induction of pluripotency appeared to be associated not with a unique mtDNA rearrangement but rather with a consistent overall readaptation of metabolic and bioenergetic pathways to the acquired pluripotent state. Overall, our findings demonstrate the presence of various homoplasmic and heteroplasmic mtDNA mutations within human iPSCs and suggest the integrity of the mitochondrial genome as an additional aspect to be assessed before using iPSC lines for modeling or clinical applications.
We thank all colleagues in the Adjaye lab for fruitful discussion, A. Sabha and C. Vogelgesang at the microarray facility, Dr. A. Wulf-Goldenberg and M. Keil (EPO-Berlin Gmbh) for the teratoma assay, Dr. G. Thiel (Human Genetic Center of Berlin) for the karyotypic analysis, E. Jansen and Prof. C. Jakobs from VUMC for help with mass spectrometry analysis, and Dr. D. Ferrick, Dr. J. Dunn, and H. Hedeby from Seahorse Bioscience for scientific and technical support. This work was supported by the BMBF (01GN0807) and the Max Planck Society.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.