Generation and characterization of human induced pluripotent stem cells
A dermal biopsy specimen was collected after obtaining written informed consent from a healthy individual and cultured on a Petri culture dish. Fibroblasts were isolated and expanded by the outgrowth method in DMEM supplemented with 10% FBS. Fibroblasts were passaged twice and then infected for iPSC generation. Reprogramming of fibroblasts to pluripotency was performed by nonintegrating Sendai-virus-mediated transfection of the four canonical transcriptional factors (OCT4, SOX2, KLF4, and c-MYC) (CytoTune2.0 Sendai vectors; Thermo Scientific). Briefly, 3 × 105 fibroblasts were infected at a multiplicity of infection (MOI) of 5, yielding different iPSC clones generated under feeder-independent conditions on Matrigel-coated dishes (BD Biosciences). Generated hiPSCs were stained for alkaline phosphatase (AP) activity (Additional file 1: Figure S1A) and subsequently picked manually for culture and propagation. Prior to performing pluripotency assays, generated hiPSCs were tested for loss of Sendai virus transgenes by RT-PCR (Additional file 1: Figure S1B). The pluripotency of generated hiPSCs and hESCs was evaluated by qRT-PCR for expression of the endogenous pluripotency genes OCT4, SOX2, c-MYC, REX1, and NANOG (Additional file 1: Figure S1C) and pluripotency markers Oct4 and Nanog by immunostaining (Additional file 1: Figure S1D). To further assess the pluripotency of both stem cell lines used in this study, we performed a genome-wide gene expression profile assay according to the PluriTest algorithm  (Additional file 1: Figure S1E). Additionally, generated hiPSCs and hESCs were tested for markers of the three germ layers, Nestin (ectoderm), Brachyury (mesoderm), and Sox17 (endoderm), on whole embryoid bodies (EBs) by immunostaining (Additional file 1: Figure S1F) and by qRT-PCR for endoderm (SOX7), mesoderm (HAND1, ACTA2, and MYL2), and ectoderm (NESTIN and BMP4) expression markers (Additional file 1: Figure S1G). hESCs (H9) were purchased from the WiCell Research Institute, and this cell line was used as a control throughout our experiments. Before performing experiments, all cell lines were tested for mycoplasma contamination.
Human iPSCs and ESCs were cultured on Matrigel-coated (BD Biosciences) dishes in mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada). Cells were maintained at 37 °C in 5% CO2 in a humidified incubator. The culture medium was changed daily, and cells were passaged every 4–6 days (at 70% confluence) with Gentle Cell Dissociation reagent (STEMCELL Technologies).
Reverse transcription PCR and quantitative real-time PCR
Reverse transcription PCR (RT-PCR) was used for Sendai viral transgene detection in infected parental fibroblasts (ipF) and loss in hiPSCs compared to their uninfected parental cells (pF). Quantitative reverse transcription PCR (qRT-PCR) was used to assess the expression of pluripotency genes as well as genes of the three germ cell layers. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. One microgram of RNA was used for cDNA synthesis using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Gene expression was quantified by qRT-PCR analysis using 1 μl of the RT product and Power SYBR Green Master Mix (Applied Biosystems). qRT-PCR was performed in a StepOne Plus instrument (Applied Biosystems), and the gene expression levels were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. The gene expression and relative fold-change (Fc) patterns were assessed by the 2–ΔΔCt method. The primers used in this work are presented in Table 1.
Genome-wide gene expression profile
For PluriTest assays, RNA was extracted from hiPSCs and hESCs using the Stratagene Absolutely RNA kit. Total RNA (0.5 μg) was processed with an Illumina TotalPrep RNA Amplification Kit (Thermo Scientific) following the manufacturer’s instructions. The antisense RNA (aRNA) product was hybridized to the Human HT-12v4 Expression BeadChip Kit (Illumina) and run in an iSCAN system (Illumina). The raw data were uploaded to the PluriTest website (http://www.pluritest.org) and analyzed with the PluriTest algorithm .
For immunocytochemistry, hiPSCs and hESCs were fixed in 4% (vol/vol) paraformaldehyde (PFA) and subjected to immunostaining using the following primary antibodies: human Oct4 (1:400, mouse monoclonal; STEMCELL Technologies), human Nanog (1:1000, rabbit polyclonal; Abcam), human Nestin (1:1000, mouse monoclonal; STEMCELL Technologies), human Brachyury (1:20, goat polyclonal; R&D systems), and human Sox17 (1:20, goat polyclonal; R&D systems). Incubation with primary antibodies was performed overnight at 4 °C. After rinsing with Dulbecco’s phosphate-buffered saline (DPBS), goat anti-mouse Alexa-Fluor-647, donkey anti-Goat Alexa-Fluor-594, and goat anti-rabbit Alexa-Fluor-488-conjugated secondary antibodies (all from Thermo Scientific) were added, and cells were incubated for 1 hour at 37 °C. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Slides were mounted with Fluorescent mounting medium (Dako Cytomation), and microscopy was performed using imaging systems (DMi8), filter cubes, and software from Leica microsystems.
DNA and RNA analyses for nucleic acid quantification and gel electrophoresis
Genomic DNA (gDNA) from hiPSCs and hESCs was extracted using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Saint Louis, MO, USA), while total RNA was extracted using an Absolutely RNA Miniprep kit (Agilent Technologies). Prior to DNA/RNA extraction, hiPSCs and hESCs were counted, and 4 × 105 cells were processed for nucleic acid purification. DNA and RNA samples were eluted in an equal volume of elution buffer, and 1 μl of each DNA/RNA sample was used for quantification by a NanoDrop spectrophotometer (Thermo Fisher Scientific); 0.5 μg of each RNA and DNA sample were loaded onto 1% agarose gels for electrophoresis and mass quantification. Nucleic acid purification and agarose gel electrophoresis were performed in biological triplicate for each cell line tested.
For mitochondrial labeling and activity, hiPSCs and hESCs were incubated for 30 minutes at 37 °C with 100 nM MitoTracker Green FM (Thermo Fisher Scientific) diluted in growth medium (mTeSR1; STEMCELL Technologies). Fluorescence was measured with a Leica imaging system (DMi8), and the fluorescence intensity (magnification × 20) was analyzed using Leica LAS-X software. The results are presented as the mean ± standard deviation (SD) of three independent experiments.
Cell proliferation assay by CFSE
Cell proliferation assays of hiPSCs and hESCs were evaluated by the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) method. Briefly, 5 × 105 cells were labeled with 8 μM CellTrace CFSE (cell proliferation kit; Thermo Fisher Scientific) in mTeSR1 medium for 10 minutes at 37 °C. Labeling was quenched by adding cold PBS with 0.1% bovine serum albumin (BSA) to cells, followed by a 5-minute incubation on ice. Two hours later (T0) and after 4 days (T4) of culture in mTeSR1 medium, cells were harvested for CFSE fluorescence evaluation by flow cytometric analysis (BD LSRFortessa x-20). Cell proliferation was calculated by measuring the decrease in label intensity in successive daughter cell generations . The proliferation index and cell populations of parental or successive generations were calculated with Modfit LT Version 3.2 software.
Propidium iodide staining for cell cycle analysis
Analysis of the cell cycle status was performed by flow cytometry on cells labeled with propidium iodide (PI), a fluorescent intercalating agent that is used to assess the DNA content during the cell cycle. For this assay, hiPSCs and hESCs were treated by Accutase for single cell dissociation, and 5 × 105 cells were harvested in PBS and alcohol-fixed with 70% cold ethanol at 4 °C for 30 minutes. After fixation, cells were washed three times with cold PBS, spun, and treated with PBS containing 0.1% Triton, 5 μg/ml PI, and 5 μg/ml ribonuclease for 1 hour in the dark. PI-stained cells were then analyzed by flow cytometry (BD LSRFortessa x-20) for proliferation and cell cycle distribution estimation.
Karyotype analysis of hiPSCs and hESCs was performed by multiplex-fluorescence in-situ hybridization (M-Fish). Cells were treated with KaryoMAX Colcemid solution (Thermo Fisher Scientific) and processed with standard methods. Briefly, fixed cells dropped onto glass slides were hybridized with the 24XCyte Human Multicolor FISH Probe Kit (MetaSystems, Altlussheim, Germany), following the manufacturer’s instructions. Slides were denatured in 0.07 N NaOH and then rinsed in graded ethanol. Meanwhile, the probe mix was denatured in a MJ mini personal thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following program: 5 minutes at 75 °C, 30 seconds at 10 °C, and 30 minutes at 37 °C. Samples were then hybridized in a humidified chamber at 37 °C for 48 hours, followed by one wash in saline–sodium citrate (SSC) buffer for 5 minutes at 75 °C and counterstaining with DAPI. Finally, metaphases were visualized and captured using an Axio-Imager Z2 microscope. Karyotyping analysis was performed by means of ISIS software. To determine the karyotype of the hiPSCs and hESCs, 50 metaphase spreads were analyzed.
Embryoid body formation
For EB formation, hiPSCs and hESCs were dissociated into single cells by Accutase (Thermo Fisher Scientific) and cultured on an ultralow attachment plate (Corning) with mTeSR1 medium supplemented with 10 μM Rho-kinase inhibitor Y-27632 (Selleckchem) for 3 days to enable cell aggregation. After 3 days, the medium was switched to DMEM/F12 containing a 20% knockout serum replacement (KSR), 2 mM l-glutamine, 1 × 10−4 M nonessential amino acids, 1 × 10−4 M 2-mercaptoethanol, and 0.5% penicillin and streptomycin (all from Thermo Fisher Scientific). The medium was changed every other day until day 8 . After 8 days in culture as floating EBs, cell aggregates were transferred onto 0.1% gelatin-coated plates (Sigma-Aldrich) and cultured in the same medium for an additional 8 days before collecting the EBs for immunofluorescence and qRT-PCR analyses.
hPSC culture for Raman spectroscopy measurements
For Raman spectroscopy, hiPSCs and hESCs (all at passage P40) were dissociated into single cells by Accutase (Thermo Fisher Scientific), and 4 × 105 cells per cell line were seeded on CaF2 slides because of its negligible Raman signal for 24 hours to allow the cells to adhere to the CaF2 surface in mTeSR1 medium. Prior to Raman measurements, cells were fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 minutes at room temperature. After incubation in a fixative solution, cells were rinsed with DPBS and kept in distilled water for analysis to reduce the background interference derived from the culture medium.
Raman mapping and spectra preprocessing
Raman imaging was performed with an Alpha-300R microscope from Witec GmbH (Ulm, Germany) equipped with a 532-nm laser source in a backscattering configuration. The total laser power applied to the sample was set to 10 mW to avoid cell photodamage, and light was focused on the sample through a 100×/0.9 NA objective. A 600 lines/mm grating was used for frequency analysis of the backscattered light, with a spectral resolution of approximately 3.0 cm–1. For each measured cell, Raman maps were recorded using a raster scan with a step size of 400 nm, which is close to the optical resolution of the system (≈360 nm) as calculated with the Rayleigh criteria. For each pixel, we used a typical integration time of 2.0 seconds, with a spectral window ranging from 400 to 3100 cm–1. The Raman shift was previously calibrated by measuring a silicon sample and using the sharp Si-peak at 520 cm–1 as a reference. After Raman measurements, the spectra were first divided into two spectral regions: one ranging from 400 to 1800 cm–1, which is the well-known fingerprint region; and a second region ranging from 2600 to 3100 cm–1, in which the CH2 and CH3 stretching vibrations were located. The spectra collected from the surrounding area of the cells were used as background spectra and subtracted from the cell signals. Finally, all of the spectra of one map were normalized to the maximum total spectral area recorded for that specific cell, allowing a comparison of Raman maps recorded from different cells at different times.
Principal component analysis (PCA) and K-means cluster analysis (KCA) were performed on the collected datasets. To compare the results of multivariate analysis between different maps, the spectra from all the probed cells were processed altogether as one single collection, and the computed principal components (PCs) were exactly the same for all maps. The first six PCs, which comprised more than 98% of the total variance, were used to perform KCA, imposing six clusters to be addressed inside the cells (plus one cluster collecting the empty areas outside the cells). Subsequently, pseudo-color images were generated to represent the multivariate results. A specific color was assigned to each cluster and the cluster’s spatial distribution was mapped in the xy space. A custom-developed software package, Raman Tool Set, freely available online (http://ramantoolset.sourceforge.net) , was used to perform all of the spectra preprocessing steps and multivariate analysis.
All experiments were performed at least three times, each in biological replicates. Data were analyzed using GraphPad Prism 6 software, and statistical analysis was performed by Student’s t test. All values are expressed as the mean ± standard error of the mean (SEM) in all figure panels in which error bars are shown, and differences with p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.