Healthy donor-derived human iPS cells (hiPSCs, hCD34-iPSC16)18 were provided by Dr. Nico Lachmann and Dr. Thomas Moritz (Hannover Medical School, Hannover, Germany). This hiPSC line was maintained on Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat Nr. A1413302, Thermo Fisher Scientific) –coated cell culture plates in Stemflex medium with 10% Stemflex Supplement (Cat Nr. A3349401, Thermo Fisher Scientific) and 1% penicillin/streptomycin. The medium was changed every day or every second day. Cells were passaged every 5 or 6 days in 1:10 or 1:15 ratios depending on their density.
Treatment of iPS cells with FK866 or AC93253
5×104 hiPSCs /well were seeded into one well of 6 well plate, were kept in maintenance for 48 hours and then treated with different doses of FK866 (Cat Nr. F8557-25MG, Sigma-Aldrich) or AC93253 (Cat Nr. A9605-10MG, Sigma-Aldrich). The corresponding concentration of dimethylsulfoxide (DMSO, Sigma-Aldrich) was used as vehicle control. After 48 hours cells were collected for further analysis.
Flow cytometry analysis
To assess the pluripotency of iPS cells, the antibodies TRA1-60-PE (Cat Nr. MA1-023-PE, eBioscience) and SSEA4-FITC (Cat Nr. 560126, BD biosciences, BD) were used. Dead cells were excluded from the analysis by 4’,6-diamidino-2-phenylindole (DAPI, 1ug/ml) (Cat Nr. D3571, Thermo Fisher Scientific) staining. For detection of hematopoietic progenitor cells, the antibodies CD33-BV421 (Cat Nr. 366622, BioLegend, BL), CD34-PeCy7 (Cat Nr. 343615, BL), KDR-AF647 (Cat Nr. 359909, BL), CD43-PE (Cat Nr. 343204, BL), CD41a-FITC (Cat Nr. 303703, BL), CD235a-FITC (Cat Nr. 349103, BL), CD45-BV510 (Cat Nr. 103138, BL) and 7-AAD (Cat Nr. 420404, BL) were used as an ‘early-stage’ multicolor hematopoietic cells panel. For the detection of mature myeloid cells, the antibodies CD15-PE (Cat Nr. 301905, BL), CD16-FITC (Cat Nr. 302005, BL), CD14-APC-H7 (Cat Nr. 367117, BL), CD45-BV510 (Cat Nr. 103138, BL), CD33 BV-421 (Cat Nr. 366622, BL) and 7-AAD (Cat Nr. 420404, BL) were used as a ‘late-stage’ multicolor myeloid differentiation panel. Anti-mouse IgGk beads were used for compensation. Antibodies and beads for flow cytometry were purchased from BD Biosciences unless otherwise indicated. Samples were analyzed using a FACS Canto II flow cytometer (Becton-Dickinson) and FlowJo software (FLOWJO, LLC, Ashland, OR).
RNA isolation and qRT-PCR
RNA was isolated using the RNeasy mini kit (Qiagen) and cDNA was prepared from 500 ng RNA by oligo primer using the Omniscript-RT kit (Qiagen). All procedures were performed following the manufacturers’ instructions. Quantitative polymerase chain reaction (qRT-PCR) was performed using LightCycler® 480 SYBR Green I Master (Roche Applied Science). Real-time PCR detection was performed using a LightCycler 480 Real-Time PCR System (Roche Applied Science). Quantification of target genes expression was conducted in comparison to the reference GAPDH gene expression and depicted as ∆∆Ct relative to GAPDH. Primer sequences are shown in Table S1.
Lentivirus mediated gene knockdown in hiPSCs
HEK293T cells were used for lentivirus production. On the day before transfection, 8× 106 HEK293T cells were plated in each T75 flask. Cells were co-transfected with target shRNA expression vector (NAMPT shRNA in pRRL.PPT.SF.i2RFP, SIRT2 shRNA in pRRL.PPT.SF.i2GFP, p53 shRNA in pRRL.PPT.SF.i2YFP) or control vector (pRRL.PPT.SF.i2GFP, pRRL.PPT.SF.i2RFP, or pRRL.PPT.SF.i2YFP), psPAX2 packaging vector (#12260, Addgene) and pMD2.G envelope vector (#12259, Addgene) using TransIT®-LT1 Transfection Reagent (#MIR2305, Mirus Bio LLC). Oligonucleotide sequences for shRNA are available upon request. Lentivirus-containing supernatants were harvested at 48 h after transfection, passed through a 0.22 μm filter and incubated with Lenti-X™ Concentrator (#631232, Takara Clonetech) overnight at 4°C. After centrifugation, the virus pellet was resuspended with complete DMEM medium and titrated by FACS.
For knockdown of NAMPT, SIRT2, or p53, hiPS cells were seeded in 6 well plate (1 × 105 cells/well) 24h before transduction. Cells were transduced by incubation and centrifugation with lentiviral supernatant at a multiplicity of infection (MOI) 40. After 72h incubation at 37°C, transduction efficiency was quantified by qPCR and western blot.
Western blot analysis
Whole-cell lysates were obtained by lysing equal numbers of cells with 3× laemmli buffer (30% glycerol, 6% SDS, 7.5% β-Mercaptoethanol, 0.75% Bromphenol blue in 200nM Tris-HCL [pH 6.8]), which were subsequently heated at 95°C for 5min and spun down. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE healthcare life sciences). The WB membranes were blocked with 5% non-fat dry milk-TBST (10mM Tris-HCL [pH 8.0], 150mM NaCl, 0.1% Tween 20) for 1h at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing 4 times for 5min with TBST, membranes were incubated with secondary antibodies for 1 hour at room temperature. The protein bands were detected using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) or Luminata Forte Western HRP substrate (Millipore, Billerica, MA) and visualized by exposure to X-ray film (GE healthcare life sciences). The following antibodies were used: rabbit monoclonal antibody to GAPDH (Cat Nr. 2118, Cell Signaling Technology), rabbit monoclonal antibody to p21 (Cat Nr. 2947s, Cell Signaling Technology), mouse monoclonal antibody to p53 (Cat Nr. sc-126, Santa Cruz Biotechnology), rabbit monoclonal antibody to acetyl-p53 (Lys382) (Cat Nr. 2525, Cell Signaling Technology), rabbit polyclonal antibody to NAMPT (Cat Nr. PAB17046, Abnova).
Assessment of cell proliferation with Incucyte® S3 Live-Cell Analysis System
2×104 hiPSCs were seeded in Geltrex-coated cell culture plates in Stemflex medium with 10% Stemflex Supplement (Cat Nr. A3349401, Thermo Fisher Scientific). The cells were incubated in the medium supplemented with FK866 1nM/2nM, AC93253 50nM/100nM or DMSO at the corresponding concentrations in IncuCyte live cell analysis system (Essen Bio) at 37°C, 5 % CO2. The cell growth images were recorded every 6h and analyzed by IncuCyte S3 Software (Essen Bio).
Cell cycle analysis
For cell cycle analysis, iPS cells were incubated with 1mM of BrdU for 30 min and BrdU uptake was quantified using APC BrdU Flow Kit (Cat Nr. 557892, Becton-Dickinson, Franklin Lakes, NJ, USA). Samples were analyzed using a FACS Canto II flow cytometer (Becton-Dickinson) and FlowJo software (FLOWJO, LLC, USA).
Assessment of apoptosis
Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit (Cat Nr. 556547, Becton-Dickinson) following the manufacturer’s instructions. Samples were analyzed using a FACS Canto II flow cytometer (Becton-Dickinson) and FlowJo software (FLOWJO, LLC).
Embryoid body (EB)-based hematopoietic differentiation of hiPSCs
hiPS cells were kept in maintenance on Geltrex coated plates for 5 days until confluency. iPS cells were dissociated by PBS/EDTA (0.02%) for 5-7 min. EB induction was achieved via Spin EBs (20.000 cells/EB) in 96-well plates using APEL serum-free differentiation medium (Stemcell Technologies) supplemented with bFGF (20 ng/µl) and ROCK Inhibitor (R&D). After 24 hours, BMP4 (40 ng/µl) was added to the culture to induce mesodermal differentiation. After 2 days, EBs were plated on Geltrex coated 6-well-plates (10 EBs/well) in hematopoietic stem cell differentiation medium (APEL medium supplemented with 40 ng/µl VEGF, 50 ng/µlSCF and 50 ng/µl IL-3). After 3 days, medium was changed to the neutrophil differentiation medium (APEL medium supplemented with 50 ng/µlIL3and 50 ng/µl G-CSF). DMSO, FK866 (1nM and 2nM) or AC93253 (50nM and 100nM) were added to the culture medium starting at day 3 of culture. Medium with DMSO, FK866 or AC93253 was exchanged every 3 days. Hematopoietic cells appeared on day 12 - 14 of culture. They were harvested for various analyses on day 18 and 25. All cytokines were purchased from R&D System unless otherwise indicated. Cell morphology was evaluated on cytospin preparations of suspension hematopoietic cells generated on day 25 of culture. For this, 2×104 cells were centrifuged on the cytospin centrifuge at 400 rpm for 4 min. Cytospin slides were stained with Wright-Giemsa Stain using the Hema-Tek slide stainer (Ames).
Three germ layers differentiation assay
The three germ layers differentiation of hiPS cells was performed using STEMdiffTM Trilineage Differentiation Kit following the manufacturer’s instructions (Cat Nr. 05230, Stem Cell). In some experiments, cell culture medium was supplemented with FK866 1nM, AC93253 50nM, or DMSO. For ectoderm lineage, 4×105 hiPS cells per well were plated in 24-well plate on day 0. After 24 hours, the culture medium was changed from Stemflex medium to STEMdiffTM Trilineage Ectoderm Medium (Cat Nr. 05231, Stem Cell). The medium was changed daily until day seven. 1×105 hiPS cells per well for mesoderm lineage and 4×105 hiPS cells per well for endoderm lineage were plated in 24-well plate on day 2. The cells were supplemented daily with STEMdiffTM Trilineage Mesoderm Medium (Cat Nr. 05232, Stem Cell) and STEMdiffTM Trilineage Endoderm Medium (Cat Nr. 05233, Stem Cell) with inhibitors or DMSO starting from day 3 till day 7.
Cell differentiation was analyzed on day 7 using the Human Three Germ Layer 3-color Immunocytochemistry Kit according to the manufacturer’s instructions (Cat Nr. SC022, R&D Systems). Cells were fixed in PBS containing 4% paraformaldehyde (Cat Nr. 158127, Sigma-Aldrich) and blocked with PBS containing 10% normal donkey serum (Cat Nr. D9663, Sigma-Aldrich), 0.3% Triton™ X-100 (Cat Nr. 93443, Sigma-Aldrich), and 1% BSA (Cat Nr. A2058, Sigma-Aldrich). After that, cells were incubated with conjugated antibodies corresponding to the cell lineage of interest: Otx-2 for ectoderm, Brachyury for mesoderm, and GATA-4 for endoderm. After 3 hours incubation at room temperature in the dark, cells were washed and kept with PBS containing 1% BSA and DAPI (1:1000 dilution). Images were taken on ZEISS Apotome microscope.
Statistical analyses were conducted using Student’s t-test or Bootst Ratio19 Statistical significance was taken to be p< 0.05.