All pure standards were purchased from Sigma-Aldrich (St Louis, USA), except UDP-glucose that was purchased from American Custom Chemicals Corp. Ammonium fluoride, formic acid and EDTA were purchased from Sigma-Aldrich (St Louis, USA). LC/MS grade acetonitrile and water were purchased from JT Baker (Phillipsburg, USA).
300 μl of LC/MS-grade water in 1 mM HEPES and 1 mM EDTA (pH 7.2) was added to the ESC, iPSC or somatic cell pellet (~250 000 cells), vortexed for 30 s, and the sample incubated for 1-2 min in boiling water. Next, the sample was incubated for 1 min in liquid nitrogen and thawed at room temperature. The incubation in liquid nitrogen was repeated. Finally, the sample was incubated for 1 h at −20 °C, followed by 15 min centrifugation at 13 000 rpm (4 °C). The resultant supernatant was transferred to an HPLC vial.
LC-MS and MS/MS analysis
Analyses were performed using an HPLC system (1200 series, Agilent Technologies) coupled to a 6538 UHD Accurate-Mass Q-TOF (Agilent Technologies) operated in positive (ESI+) or negative (ESI−) electrospray ionization mode. Vials containing extracted metabolites were kept at −20 °C prior to LC/MS analysis. ESCs, iPSCs and somatic cells extractions were separated using a Cogent Bidentate C18: 4 μm, 100 Å, 150 mm × 2.1 mm ID (Cat No. 40018-15P-2), or a Waters XBridge C18: 3.5 μm, 135Å, 150 mm × 1.0 mm ID (Part No. 186003128) column when the instrument was operated in positive or negative ionization mode, respectively. The solvent system in positive ionization mode was: A = 0.1% formic acid in water, and B = 0.1% formic acid in acetonitrile. When the instrument was operated in negative ionization mode, the solvent system was: A = 1 mM ammonium fluoride in water, B = acetonitrile 30
. The linear gradient elution used started at 100% A (time 0-5 min), and finished at 100% B (35-40 min). The injection volume was 8 μl. ESI conditions were: gas temperature 325 °C, drying gas 11 l/min, nebulizer 30 psig, fragmentor 120 V and skimmer 65 V. The instrument was set to acquire over the m/z
range 80-1 000 with an acquisition rate of 1.3 spectra/s. MS/MS was performed in targeted mode and the instrument was set to acquire over the m/z
range 50-1 000, with a default iso width (the width-half maximum of the quadrupole mass bandpass used during MS/MS precursor isolation) of 4 m/z
. The collision energy was fixed at 20 V.
Metabolomic data processing
LC/MS data from the ESC, iPSC and somatic cell extractions (ESI+ and ESI− modes) were processed using the XCMS software (version 1.24.1) to detect and align features. XCMS analysis of these data provided a matrix containing the retention time, m/z value and intensity of each feature for every sample described above. Statistically significant differences (P-value < 0.01) are ranked using univariate t-test in order to obtain quantitative information between cell populations. In addition, using the statistical software R, each row of feature intensities was normalized such that the highest value was one. A two-dimensional representation of this matrix was calculated using MDS as implemented within the software R.
Metabolite database searching
Cell culture and cell sample preparation
Human neonatal keratinocytes (Lonza), fibroblasts (IMR90, BJ, from ATCC) and HUVECs (Lonza) were grown according to manufacturer's recommendations. 293T cells were cultured in DMEM (Invitrogen) containing 10% fetal calf serum. Human H1 (WA01) or H9 (WA09) ESC lines (Wicell) and Hues6 (Harvard), and all iPSC lines were cultured as previously described in mTeSR1 before metabolite extraction 16, 31
. For cell sample preparation, keratinocytes, fibroblasts, ESCs and iPSCs (individual iPSC and ESC colonies were mechanically picked) were individualized using TrypLE (Invitrogen) according to manufacturer's recommendations. Equivalent numbers of cells were pelleted, washed 1× in PBS, and stored in −80 °C until metabolites were extracted as described.
iPSC generation and reprogramming experiments
Keratinocytes or fibroblasts were infected with equivalent ratios of retroviruses encoding KLF-4, OCT4, SOX2 and c-MYC (KOSM) by spinfection at 800× g
for 1 h at RT in the presence of polybrene (8 μg/ml). Cells were replated onto MEFs (Millipore) in their respective medias, and switched to ESC medium for iPSC colony formation. Resulting iPSC colonies were manually picked for iPSC line derivation (~14 days after infection for keratinocytes, and ~3-4 weeks after infection for fibroblasts). For reprogramming experiments, dFibOCT4GFP
or keratinocytes were KOSM-infected as described. Reprogramming efficiencies were then determined by calculating the number of GFP-positive, or Nanog-positive colonies, respectively, in metabolite treated conditions (2-DG, Sigma; F6P, Sigma; arachidonic acid, Cayman Chemicals; S-adenosyl methionine, New England Biolabs) relative to controls. Metabolite concentrations for use in reprogramming experiments were determined empirically by examining somatic cell survival by TUNEL staining.
Plasmids and virus preparation
The following moloney-based retroviral vectors were obtained from Addgene: pMX-OCT4, pMX-SOX2, pMX-KLF-4 and pMX-c-MYC (plasmids 17217, 17218, 17219 and 17220, respectively). Packaging plasmids (pCMV-gag-pol-PA and pCMV-VSVg) were kindly provided by Dr Gerald Pao (Laboratory of Genetics, The Salk Institute, La Jolla, CA). Retrovirus was collected 24 h following cotransfection of plasmids in 293T cells using Lipofectamine (Invitrogen) in accordance with manufacturer's recommendations.
Extracellular flux bioenergetic assay
Assays were performed in accordance with manufacturer's instructions (Seahorse Bioscience). Briefly, cells were seeded in extracellular flux 96-well cell culture microplates (Seahorse Bioscience) in 80 μl of corresponding cell growth medium (ESCs and iPSCs were grown in mTeSR1, Stem Cell Technologies) and then incubated at 37 °C/5% CO2 for ~24 h. Assays were initiated by replacing the cell growth medium from each well and replacing it with 200 μl of assay medium. The microplates were incubated at 37 °C for 60 min to equilibrate the temperature and pH of the media before measurement. A Seahorse Bioscience instrument (model XF96) was used to measure the rate of change of dissolved O2 and pH in the media. OCR and ECAR were measured simultaneosly for ~3 min in repeated cycles, to obtain a basal average. All OCR and ECAR values were normalized by cell number.
Genomic DNA was extracted using the ALLPrep DNA/RNA Mini kit (Qiagen). The bisulfite conversion and capture reactions was carried out on each gDNA sample of somatic or iPSCs 10, 32
using previously established protocols 18
. Briefly, DNA was bisulfite converted using the Zymo Methyl-Direct Bisulfite Conversion kit. Bisulfite-converted DNA was then captured using specially designed padlock probe oligonucleotides, resulting in a circular DNA library of targeted CpG sites. The padlock capture and DNA sequencing was carried out using a previously described protocol 33
. Bisulfite converted data was processed as previously described 18
. Heatmaps and dendrograms were created from the Pearson's correlation matrices of (1) the relative change in methylation level between each iPSC line and its somatic progenitor, and (2) the absolute methylation level at each site in each iPSC line.
Identification of differentially methylated sites in oxidative phosphorylation and in glycolysis
To identify sites showing a change in methylation after reprogramming, a Chi-squared test with Yates' correction was carried out on each CpG site characterized in each iPSC line and corresponding paired-somatic cell line. The Benjamini-Hochberg method was used to correct for multiple testing errors; the false discovery rate was set at 1%. This resulted in a set of differentially methylated sites (DMSs) for each iPSC line; at each site, the methylation level was statistically significantly different from the somatic progenitor line. The DMS list was uploaded to GREAT (http://great.stanford.edu
) to find possible gene to DMS interactions based on distance from transcription start sites and curated regulatory regions of genes. Genes were then annotated by using the KEGG database for involvement in oxidative phosphorylation and in glycolysis.
Results are shown as mean values ± SEM. Statistics were performed using either an unpaired two-tailed Student's t-test or one-way ANOVA with a post-hoc test as appropriate. P-values < 0.05 were considered statistically significant.