All experiments involving animals were approved by the Institutional Animal Care and Use Committees (IACUCs) from the Magee-Womens Research Institute and the University of Pittsburgh and experiments involving embryonic stem cells were approved by the University of Pittsburgh Human Stem Cell Research Oversight (hSCRO) committee.
NhpESC cell lines were a generous gift from Gerald Schatten (U. Pittsburgh). NhpESC lines were previously described (, personal communication). Primary adult non-human primate lung fibroblasts were obtained during necropsy by taking tissue biopsies. Tissues were minced and grown in DMEM with 15% serum, 1% penicillin/streptomycin, and 2 mM L-glutamine.
NhpESCs were cultured in 80% KO-DMEM (Invitrogen, Carlsbad, CA), 20% Knockout Serum Replacement (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), and 4 ng/ml basic human recombinant FGF (Invitrogen). NhpESCs were grown on inactivated mouse embryonic feeder cells (MEF’s) and manually passaged weekly. Media was replaced every 24 hours .
Pluripotency marker expression
NhpESCs were fully characterized to demonstrate pluripotency using a teratoma assay . To ensure nhpESCs maintained a pluripotent phenotype throughout the experiments, cells were stained for the positive pluripotency markers: Oct-4, SSEA-4, Tra 1–80 and Tra 1–61, as well as the negative nhpESC marker SSEA-1. Immunocytochemistry was performed as follows: Culture dishes containing undifferentiated colonies were fixed by addition of either 100% methanol at -20°C for 15 min, or 2% paraformaldehyde in PBS for 40 minutes followed by a 15 min wash in PBS + 1% Triton X-100 (PBS-Tx, Sigma, St. Louis MO). After fixation, non-specific binding of the primary and secondary antibodies was blocked by 30 minutes incubation in PBS with 0.5% goat serum. Primary antibodies were diluted in PBS-Tx and incubated on the coverslips for 40 min at 37°C in a humidified chamber, with the exception of Oct-4 which was incubated overnight at 4°C. Primary antibodies were detected with fluorescently labeled appropriate secondary antibodies. DNA was detected with 5 μM TOTO-3 (Molecular Probes, Eugene OR). Coverslips were inverted onto slides and mounted in Vectashield anti-fade medium (Vector Labs, Burlingame, NH) to prevent photobleaching .
In Vitro differentiation
Colonies were manually passaged and dissociated into small clusters. They were cultured in non-adherent dishes to form embryoid bodies in differentiation medium (80% KO-DMEM, 1 mM L-glutamine, 20% FBS, 1% non-essential amino acids). After 4 days in suspension, aggregates were transferred onto gelatin coated culture dishes and cultured for an additional 9 days in differentiation medium. Outgrowth cultures were manually passaged by scraping cells with a differentiated phenotype from the periphery of the colonies, and these differentiated cells were placed in a new culture plate in differentiation medium. After the first passage, cells were grown in standard fibroblast media (90% KO-DMEM, 10% FBS, 1% non-essential amino acids, 2 mM L-glutamine) and passaged at 80% confluency using trypsin. Cells were banked at each passage and were cultured until they stopped growing or through passage 10. Experimental groups were treated with 100 nM nicotine starting on day 1 of the differentiation protocol.
PCR for nAChR
RT-PCR for nAChR was done using primer sequences that have been previously published . All primers span introns and do not amplify DNA. GAPDH or actin was always used as a positive control for RNA integrity. Oligo dT12-18 (Invitrogen) was annealed to 1 μg total RNA and reverse transcribed with Superscript II (Invitrogen). The reaction contained 1 μg RNA, 500 ng Oligo dT12-18, 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each dNTP, 200 U superscript. Briefly, total RNA was incubated with oligo dT12-18 at 70.0°C for 10 min. The cDNA produced was then used as a template for PCR using specific primers. PCR amplification was performed in a 20 μl reaction containing 2 μl of the RT reaction, Taq DNA polymerase (Perkin Elmer), 1X PCR buffer, 1.5 mM MgCl2, 1 mM each dNTP and 1 μM primer. PCR was carried out in a Perkin-Elmer 9700 Thermocycler with 2 min, 95.0°C denaturation, followed by 30 cycles of 94.0°C for 30 s, 55.0-62.0°C (see ) for 30 s and 72.0°C for 30 s. Final extension was at 72.0°C for 5 min. 10 μl of each reaction was run on a 1% TBE gel for analysis. β2 and δ subunits were detected using nested PCR. Primary PCR reactions were carried out as described above. 2 μl of the primary reaction was used as the template for the secondary PCR reaction/second round PCR. Thirty rounds of PCR were carried out.
Differentiated fibroblasts were tested for their ability to form teratomas in the testes of mice. The analysis was done at the MWRIF Transgenic Core Facility according to their standard methods [12, 22, 35–38]. Pathology on the testes was performed by the MWRIF Histology Research Core Facility.
Gene expression analysis
Gene expression analysis was performed by the DNA Analysis group at the University of Pittsburgh Genomics and Proteomic Core Laboratory using cell pellets provided by D. Carlisle’s laboratory as described herein. RNA was isolated from in vitro differentiated fibroblasts from three different nhpESC lines, each differentiated in the presence and absence of nicotine: nhpESC2706, nhpESC3106 and nhpESC4706. Three technical replicates were done with each cell line and condition, and cultures with and without nicotine were matched for passage number after differentiation. RNA Isolation: RNA was purified using a modified Trizol extraction method (Invitrogen, Carlsbad, CA). Briefly, suspended cells were extracted in 1 mL of Trizol with the addition of 200 μg of GlycoBlue (Ambion Inc, Austin, TX) added to each sample as a nucleotide carrier. After aqueous phase separation, the samples were incubated overnight at 20°C in 500 μl of isopropanol (100%) to precipitate the RNA. The RNA was then pelleted by centrifugation (14,000 g × 15 min), washed in 1 mL of 75% ethanol, and resuspended in 20 μl nuclease-free water at 45°C for 5 minutes. The RNA concentration and quality was evaluated with criteria for inclusion in subsequent in vitro transcription (IVT) assays comprising spectrophotometric absorption ratio of 260/280 >1.8 (NanoDrop, Wilmington, DE) and a RIN value of >8.0 via electrophoretic analysis (Agilent Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA). Affymetrix Eukaryotic Target Preparation and Hybridization: In vitro transcription was performed using the Ambion Message Amp II-Biotin Enhanced Assay protocol starting with 100 ng of purified total RNA. Confirmation of cRNA diversity was obtained using the Bioanalyzer 2100 to generate an electrophoretogram for each IVT reaction regarding sample yield, integrity, and size diversity against a Universal Human Reference RNA (Stratagene, La Jolla, CA). Fifteen micrograms of purified, biotin labeled cRNA was fragmented and hybridized on Rhesus Macaque Whole Genome Arrays (Affymetrix Corp., Santa Clara, CA) for 18 hours. Washing, staining and scanning of arrays were performed on the Fluidics Station 450 and Scanner 3000 immediately after completion of hybridization. Microarray data was processed using GeneChip Operating Software (GCOS, Affymetrix) with signal intensity calculated by Microarray Suite version 5.0 (MAS 5.0).
Differential gene expression analysis was performed in consultation with the University of Pittsburgh Genomics Analysis Services using BRB Array Tools from NCI (http://linus.nci.nih.gov/BRB-ArrayTools.html), and genes were selected at p < 0.001 (Additional file 1: Table S1). Statistical analysis of qPCR and immunoblotting was done by t-test, and analysis of slope for doubling time was done using a two-factor ANOVA for interaction on log-transformed cell counts.
Cells were pelleted by centrifugation at 200 × g for 5 min. RNA was isolated using Trizol (Invitrogen). Briefly, 1 ml of Trizol was added per cell culture dish and vortexed to lyse cells to homogeneity. 200 μl of chloroform (Sigma) was added followed by mixing and centrifugation for five min at 25,000 × g. The RNA containing supernatant was removed and the RNA pelleted using 600 μl of 100% isopropanol added to the supernatant and incubated at -20°C for at least 4 hrs. The sample was centrifuged twice at 13000 × g for 30 min at 4°C, with an ethanol wash in between, followed by air-drying. The RNA pellet was reconstituted in nuclease free water and treated with 1 μl DNase I for 30 min at 37°C. cDNA was prepared using the Improm-II Reverse Transcription System (Promega, Madison WI), according to manufacturer’s directions. Validated primer sets were purchased from Applied Biosystems and were used on an ABI 7900HT real-time PCR machine. Real-time data was analyzed and fold change was calculated using the comparative CT method with either beta-actin or GADPH as the internal control .
Protein isolation and immunoblotting
Proliferating cells at passage 8, 9, or 13 were scraped from flasks in ice cold PBS/Phosphatase Inhibitors. Cell pellets were lysed in RIPA Buffer supplemented with a Protease Inhibitor Cocktail (Roche). The lysate was centrifuged for 20 minutes at 14,000 × g and 4°C in a precooled microcentrifuge. Protein concentrations of the clarified lysates were quantitated using the BCA Assay Kit (Thermo Scientific) and a BSA standard curve following the manufacturer’s instructions. Protein samples were stored at -80°C. Samples for Western blot analysis were diluted in 4 × Laemmli buffer (BioRad) supplemented with 10% β-mercaptoethanol to a concentration of 10 μg of protein in 20 μL. Samples were heated at 55°C for 5 minutes and were separated on 10% polyacrylamide gels by SDS-PAGE and transferred to Immobilon-FL PVDF membranes (Millipore) by BioRad wet transfer. The membranes were blocked with LiCor Odyssey blocking buffer for 1 hour at room temperature and were then probed with a primary N-myc antibody (NCM II 100, Millipore) diluted 1:100 in LiCor Odyssey blocking buffer with 0.05% Tween. Membranes were washed three times for 15 minutes each in PBS supplemented with 0.05% Tween (TBST) and were subsequently probed with goat anti-mouse IR-Dye 800cw labeled secondary antibody diluted 1:30000 in LiCor Odyssey blocking buffer. Washes were repeated after labeling with secondary antibody three times in PBST and then once in PBS. Membranes were imaged using LiCor scanner. Membranes were stripped using LiCor NewBlot stripping buffer for 15 minutes at room temperature. Membranes were reprobed with mouse anti-β-actin diluted 1:10000 in LiCor Odyssey blocking buffer with 0.05% Tween followed by washes and probing with the LiCor secondary antibody, then washed and imaged as described above.
Analysis of culture doubling time
For growth analysis, cells were plated in triplicate in 6-well plates (Corning Life Sciences, Corning, NY) at a concentration of 2 × 105 cells/well. After 2, 3, 4, 5, 6, and 7 days, cells were trypsinized and counted using the Countess Automated Cell Counter (Invitrogen). Cell counts were analyzed by logistic regression and doubling times were calculated using http://www.doubling-time.com/compute.php.