KLF4, OCT3/4, SOX2
, and c-MYC
were amplified by polymerase chain reaction (PCR) using Phusion™ High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA), and they were in-frame linked by E2A, T2A, and P2A sequence [11
], respectively, as a single open reading frame (ORF) designated as KOSM
. The KOSM
fusion gene was then cloned into the lentivirus vector pLentG, and the resulting plasmid was designated as pLentG-KOSM. The complete sequence will be provided upon request.
Transfection and western blot analysis
One μg of pLentG-KOSM was transfected into 293T cells in a 6-well plate by Fugene HD (Roche). Forty-eight hours after transfection, the cells were washed with cold PBS buffer and lysed directly with RIPA lysis buffer, supplemented with protease inhibitor cocktail (Sigma). The cell lysates were separated by electrophoresis on 12% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Pierce). The blot was blocked with TBST (20 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.05% Tween-20) containing 5% non-fat milk, and then incubated with primary antibody solution at 4 °C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for one hour at room temperature. Signals were detected with the Immobilon Western Chemiluminescent HRP substrate (Pierce). Primary antibodies were: anti-OCT3/4 (1:2 000, #SC-5279, Santa Cruz, CA), anti-SOX2 (1:2 000, #2748s, Cell Signaling, Danvers, MA), anti-KLF4 (1:2 000, #SC-20691, Santa Cruz, CA), anti-c-MYC (1:2 000, #9402, Cell Signaling, Danvers, MA), anti-β-Actin (1:5 000, #A300-491A, Bethyl Laboratories, Montgomery, TX), anti-mouse IgG-HRP (1:5 000, #1858413, Pierce, Rockford, IL), anti-rabbit IgG-HRP (1:2 000, #1858415, Pierce, Rockford, IL).
Preparation of mouse embryo fibroblasts (MEFs)
The embryos were harvested from pregnant female (C57/BL6 background) mice on day 14~16 of gestation, and were removed from the extraembryonic membranes while the embryos were in a petri dish with PBS. The heads and all internal organs were removed and the embryo carcasses were washed with PBS buffer. The prepared individual embryo were transferred into a 6-well plate on ice with 0.25% trypsin, minced with scissors, and then left in cold room overnight. Twelve hours later, the minced embryoes were moved to a 37 °C bath for 20 min to activate trypsin, followed by addition of DMEM medium containing 10% FBS (heat-inactivated). The embryo pieces were vigorously disrupted until cloudy, and then spun down and aspirated off the medium. The MEFs were cultured in fresh DMEM-F12 cell culture medium at 37 °C.
Lentivirus production and generation of iPS cells from MEFs
The 293T cells were plated at 8 × 105 cells per 60 mm dish and incubated overnight. The next day, the cells were transfected with a mixture of DNA containing 2 μg of pLentG-KOSM, 1 μg of pCMV-VSVG, and 1.5 μg of psPAX2 (Addgene) by Fugene HD (Roche), according to the manufacturer’s instruction. Twenty-four hours after transfection, the supernatant of transfected cells was collected and filtered through a 0.22 μm pore-size filter. For virus infection, MEFs (passage 3) were seeded in a 6-well plate at 2 × 104 cells per well at one day before transduction. The medium was replaced with virus-containing supernatant supplemented with 8 μg/ml polybrene (Sigma), and centrifuged at 900 g for 1 h. The cells were infected twice and incubated with fresh DMEM-F12 medium.
At day 3 post-infection, the medium was switched to the serum-free ESGRO medium (Millipore) and changed daily until induced colonies were picked up at day 15 post-infection. GFP ratio was determined by flow cytometry at day 3 after virus infection. Individual ES cell-like cell colony was monitored for GFP marker expression during reprogramming.
Immunofluorescence staining, Immunocytochemistry and alkaline phosphatase staining
For immunofluorescence staining of cells, cells were fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, the cells were treated with PBS containing 1% bovine serum albumin (BSA), and 0.1% Triton X-100 for one hour at room temperature, followed by incubation with primary and second antibodies.
For immunocytochemistry immunofluorescence staining of tissues, 5 μm paraffin-embedded sections were prepared from teratoma and mouse embryos, and incubated in citrate buffer (pH 6.0) at 92 °C for 20 min. The sections were then washed three times with phosphate-buffered saline (PBS). Staining of teratomas was performed with the avidin/biotin blocking kit and the M.O.M. Peroxidase Kit (Vector Laboratories) according to the manufacturer’s guidelines. Sections of mouse embryos were stained with anti-GFP antibody (1:400, 3E6, A-11120, Invitrogen).
Following antibodies were used in this study: anti-OCT3/4 (1:50, #sc-5279, Santa Cruz), anti-SOX2 (1:100, #2748s, Cell Signaling), anti-NANOG (1:200, A300-398A-1, Bethyl Laboratories, Inc), anti-βIII-Tubulin (1:100, #CBL412, Millipore), anti-α-SMA (1:100, #CBL171, Upstate), anti-CK18 (1:500, #C-04, ab668, Abcam), anti-Albumin (1:50, #A90-134A, Bethyl Laboratories), or anti-GFP (1:400, 3E6, #A-11120, Invitrogen). Secondary antibodies: Texas Red-conjugated goat anti-mouse IgG (1: 100, #T-6390, Invitrogen) or FITC-conjugated goat anti-rabbit IgG (1:100, #81-6111, Invitrogen).
Alkaline phosphatase staining was performed by using the alkaline phosphatase detection kit (#SCR004, Millipore).
RNA extraction and RT-PCR
Total RNA was extracted by using Trizol reagent (Invitrogen) and treated with DNase I (Roche) to remove genomic DNA contamination. One μg of total RNA was used to synthesize cDNA by using qScrip cDNA supermix kit (Quanta Biosciences) and dT20 primer, according to the manufacturer’s instructions. PCR was performed with gene-specific primers. The list of primers is included in the Supplementary information, Table S1
The cells were cultured for 24 h in a CO2 incubator, and treated with colcemid (0.07 μg/ml, final concentration) for four hours before harvesting. The cells were washed with PBS and then trypsin lysed and transferred into 15 ml tubes. The cells were centrifuged for 10 min at 500 g and the supernatant was removed and resuspended with 10 ml KCl solution (75 mM). The cell mixtures were incubated for 30 min in 37 °C water bath and then fixed by adding 2 ml fixative solution (methanol/acetic acid 3:1). The fixed cells were washed at least two times with 10 ml of fixative solution before being applied onto chilled slides. The slides with chromosomes were dried and treated with 0.0025% trypsin for 5 min and stained with Giemsa (1:10) for 5 to 10 min.
Microarray and array analysis
For microarray experiment, RNA concentration and purity were determined by measurement of A 260, A260/A280 and A260/A230ratios using a NanoDrop-1000 Spectrophotometer (Nano-Drop Technologies). RNA quality was checked by the Agilent 2100 Bioanalyzer (Agilent Technologies). All RNA samples had high integrity (RIN > 9.9) and showed no DNA contamination. For each sample, cDNA was generated from 600 ng of total RNA with Agilent Quick Amp kit (Agilent Technologies) according to manufacturer. cDNA was then amplified and labeled with cyanine 3-CTP using Agilent Quick Amp kit. Approximately 1.7 μg of cyanine 3-labeled cRNA was fragmented and hybridized to the Agilent Whole Mouse Genome Oligo Microarray 4×44k at 65 °C for 17 h in agilent hybridization oven, according to manufacturer’s recommendation. Each sample was hybridized with 2 arrays in technical replicates. Hybridized arrays were washed and scanned on a GenePix 4000B scanner (Axon Instruments) using GenePix Pro 6.0 software (Axon Instruments) at 5 μm resolution.
The raw data were normalized (quantile normalization and log2 transformed) and filtered with Partek software (Partek) after extracting them with Feature Extraction software 9.5.1 (Agilent). ‘Fold change’ or ‘P value’ were used for identification of differently expressed genes. Thresholds for selecting significant genes were set at 2 ≤ relative fold difference ≤ −2, or P value of 0.001. Genes that met the criteria simultaneously were considered as significant changes. Pathway analysis was done with Ingenuity Pathway Analysis Software (Ingenuity Systems). The data include significant functions and significant pathways.
Calculation of reprogramming efficiency
We quantified the infected MEFs (GFP+ cells) at day 3 post-infection. We also counted the total ES cell-like cell colonies, AP+ colonies, Oct4+ and Sox2+ colonies. The total number of true pluripotent iPS cell colonies was determined based on the results of in vitro differentiation experiment.
In vitro differentiation of iPS cells
For embryoid bodies (EBs) formation, the induced colonies were expanded and were harvested by treating with Accutase (#SCR005, Millipore). The clumps of the cells were transferred to ultra-low 6-well plate in the ES cell medium (20% FBS, 2mM L-glutamine, 1 × 10−4 M nonessential amino acids, 1 × 10−4 M 2-mercaptoethanol, penicillin, and streptomycin, without LIF). The medium was changed every other day.
After 9 days of suspension in culture, the EBs were transferred to gelatin-coated plate and cultured in the same medium for another 7-9 days. For neural differentiation, all-trans retinoic acid (1 μM) was added into the medium at day 5 after suspension in culture and continued for 2 days. At day 7, the EBs were transferred to cell culture plate and cultured in the ES cell medium for another 6-7 days.
Five million iPS cells were collected from MEF feeder layers by collagenase IV and were resuspended in a mixture of DMEM culture medium and Matrigel (ratio of 1:1). The cell mixtures were injected subcutaneously into flanks of syngeneic mice. Tumors were dissected from mice 5-8 weeks after injection and paraffin sections were processed and stained with haematoxylin and eosin.
Generation and histological analysis of chimeras
Blastocysts were obtained through mating of female BDF1 (primed with hormone) and male BDF1 or C57BL/6J mice. Chimeras were produced by injecting iPS cells labeled with an EGFP expressing lentivirus into blastocysts, followed by implantation into pseudopregnant ICR mice. Chimeric embryos were dissected 10.5 days after injection and fixed. Paraffin-embedded sections (5 μm) were prepared from embryos and stained with anti-GFP antibody (1:400, 3E6, A-11120, Invitrogen).
Determination of lentiviral integration sites by inverse PCR
Lentiviral integration sites in iPS cell colonies were determined by inverse PCR according to the previous report [12
]. Briefly, Genomic DNA (100 ng) was isolated from iPS cells using the Blood & Cell Culture Mini Kit (QIAGEN) and completely digested with Taq
I restriction enzyme (clone #1, #3 – #8) or Tsp509
I (clone #2) for 12 h at 65 °C. The fragments containing the 3’LTR with flanking DNA were purified using the QIAquick Nucleotide Removal Kit and self-ligated with T4 DNA ligase (10 unit) in 100 μl volume at 15 °C overnight. The self-ligated DNA was amplified by first PCR with the primers 3’LTR-1F (5′- TGGATGGTGCTACAAGCTAGTACCAGTTGAG) and 3’LTR-1R (5′-GGTCAGTGGATATCTGATCCCTG). Nested PCR was then carried out on 2 μl of the first PCR product with the primers 3’LTR-2F (5′-AGCCAATGAAGGAGAGAACACCCGCTTGTTACAC) and 3’LTR-2R (5′-GTGGTAGATCCACAGATCAAGGATATCTTG). The resulting PCR products were run on a 2% gel.