Cell culture and vectors
Oct4-GFP MEFs were derived from mice carrying an IRES–EGFP fusion cassette downstream of the stop codon of pou5f1 (Jackson lab, Stock#008214) at E13.5. MEFs were cultured in DMEM (Invitrogen, 11995-065) with 10% FBS (Invitrogen) plus glutamine and NEAA. Only MEFs at passage 0–4 were used for reprogramming. pMX-Oct4, -Sox2, -Klf4 and -c-Myc were purchased from Addgene. Mouse AurkA was cloned into pMX. The human AurkA D274A mutant retroviral vector was purchased from Addgene. To generate retrovirus, Plat-E cells were seeded in 10-cm plates, and the next day cells were transfected with 9 μg of pMX-Oct4, -Sox2, -Klf4 and -c-Myc using Lipofectamine (Invitrogen, 18324-012) with PLUS reagent (Invitrogen, 11514-015). Viruses were collected 2 days later and combined. For reprogramming, MEFs were seeded in 12-well plates and the next day they were transduced with 4F virus using 4 μg ml−1 polybrene. One day later, the medium was replaced with fresh MEF medium, and 3 days later the medium was changed to mESC culture medium supplemented with LIF (Millipore, ESG1107). GFP+ colonies were picked on day 14 post-transduction, and expanded clones were cultured in DMEM with 15% FBS (Hyclone) plus LIF, thioglycerol, glutamine and NEAA. Irradiated CF1 MEFs served as feeder layers to culture mESC cells and derived iPSC clones.
Kinase library screening
A kinase library of 244 compounds was obtained from the chemical screening facility at the Sanford-Burnham Medical Research Institute. The library was purchased from Calbiochem (Library 1: 80 compounds, catalogue # 539744-1EA; Library 2: 80 compounds, catalogue # 539745-1EA; Library 3: 84 compounds, catalogue # 539746-1EA). All compounds are well-characterized protein kinase inhibitors.
Compounds were diluted to 2 mM in 96-well plates. 4F-transduced cells were seeded into gelatin-coated plates (3,000 cells per well). Inhibitors were added at the indicated final concentrations every other day until day 13. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and the number of Oct4-GFP+ colonies was directly counted under a microscope. Cells were then stained with Vector red AP substrate kit I (Vector laboratories, SK5100).
siRNA transfection of MEFs
siRNAs were purchased from Dharmacon and diluted in Opti-MEM (Invitrogen, 11058-021) to the desired final concentration. Lipofectamine 2000 (Invitrogen, 11668-019) was added to the mix at 2 μl per well in 12-well plates, which were incubated for 20 min at RT. For 12-well transfections, 80 μl of the siRNA/lipid mixture and 320 μl Opti-MEM was added to each well. Three hours later, 0.8 ml of the virus mixture (for iPSCs) or fresh medium was added to each well and the medium was changed to fresh MEF medium the next day. siRNAs were transfected twice during reprogramming (on days 0 and 5 post-4F transduction).
Total cell lysates were prepared by incubating cells in MPER buffer (Pierce, 78503) on ice for 20 min followed by centrifugation at 13,000 r.p.m. for 10 min. Equal volumes of lysate were resolved on 10% SDS–polyacrylamide gel electrophoresis gels, and the proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, 1620177) using the semi-dry system (Bio-Rad). Membranes were blocked with 5% milk in Tris-buffered Saline with Tween 20 (TBST). for at least 1 h at RT or overnight at 4 °C. Blots were then incubated with the following antibodies: anti-mNanog (R&D Systems, AF2729, 1:400), anti-h/mSSEA1 (R&D Systems, MAB2156, 1:400), anti-actin (Thermo Scientific, MS1295P0, 1:5,000), anti-AFP (Abcam, ab7751, 1:400), anti-β III tubulin (R&D Systems, MAB1368, 1:400), anti-α actin (Sigma, A7811, 1:400), anti-mAurkA (Bethyl Labs, A300-072A, 1:3,000), anti-hAurkA (Bethyl Labs, A300-071A, 1:1,000), anti-total-GSK3β (Cell Signaling Technology, 9315S, 1:1,000), anti-phospho-GSK3β (Ser9) (Cell Signaling Technology, 9323S, 1:1,000), anti-total Akt (Cell Signaling Technology, 9272S, 1:1,000) and anti-phospho-Akt (Ser473) (Cell Signaling Technology, 9271S, 1:1,000).
Quantitative reverse transcription–PCR
Total RNA was extracted using Trizol (Invitrogen). After extraction, 1 μg total RNA was used for reverse transcription using Superscript II (Invitrogen). Quantitative PCR was performed using a Roche LightCycler480 II (Roche) and the SYBR green mixture from Abgene (Ab-4166). Gene primers are listed in Supplementary Table S2
Cells were washed twice with PBS and fixed with 4% paraformaldehyde at RT for 20 min. Fixed cells were permeabilized with 0.1% Triton X-100 for 5 min. Cells were then blocked in 5% BSA/PBS containing 0.1% Triton X-100 for 1 h at RT. Primary antibodies were diluted between 1:100 and 1:400 in 2.5% BSA/PBS containing 0.1% Triton X-100, according to the manufacturer's suggestion. Cells were stained with primary antibody for 1 h and then washed three times with PBS. Secondary antibodies were diluted 1:400 and incubated with cells for 45 min at RT.
EB formation and differentiation assay
iPSCs were trypsinized into a single-cell suspension and the hanging drop method was used to generate EBs. Each drop consisted of 4,000 iPSCs in 20 μl EB differentiation medium. EBs were cultured in hanging drops for 3 days before being reseeded onto gelatin-coated plates. After reseeding, cells were further cultured until day 14, when beating areas could be identified.
Teratoma formation and chimera generation
iPSCs were trypsinized and resuspended at 1×107 cells per ml. Athymic nude mice were anaesthetized with Avertin, and ~150 μl of the cell suspension was injected into each mouse. Mice were examined for tumours every week for 3–4 weeks. Tumours were collected and fixed in zinc formalin solution for 24 h at RT before being paraffin embedded and stained with hematoxylin and eosin. To test the capacity of derived iPSC clones to contribute to chimeras, iPSCs were injected into C57BL/6J-Tyr(C-2J)/J (albino) blastocysts. Generally, each blastocyst received 12–18 iPSCs. Imprinting control region (ICR)-recipient females were used for embryo transfer. Donor iPSCs confer agouti or black coat colour. All animal work and use of animals complied with institutional regulations.
mRNA microarray analysis
Total RNA was extracted from derived iPSCs using Trizol. mRNA microarray analysis was carried out by the microarray facility at the Sanford-Burnham Medical Research Institute. ArrayExpress accession: E-MTAB-1188. A scatter plot was used to compare the genome-wide mRNA expression profiles of iPSCs, MEFs and mESCs.
Cell proliferation assay
MEFs were seeded at 3,000 cells per well in 96-well plates and transduced with 4F virus for 3 days. Cells were treated with inhibitors at 0.5 μM. Proliferation was measured every other day by incubating cells with mESC medium containing CellTiter 96 Aqueous One solution (Promega, G3580) for 1 h at 37 °C. Absorbance at 490 nm was measured using a plate reader, and relative proliferation curves were constructed using the absorbance signal from day 3 post-4F transduction as a reference.
Cell cycle analysis
Control or 4F-infected MEFs were treated with inhibitors for 2 days and then collected, trypsinized and fixed in 75% ethanol overnight. Cells were centrifuged at 1,000 r.p.m. for 5 min, washed once with PBS and incubated with propidium iodide staining solution for at least 30 min at RT before flow cytometric analysis. Approximately 20,000 events were collected per sample and cell cycle data were modelled using ModFit software.