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 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 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.
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 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).
Alkaline phosphatase staining
iPS cells in cell culture dishes were washed once with PBS, fixed with 4% PFA (Cat Nr. P6148-500G, Sigma-Aldrich) for 2 minutes at room temperature and washed twice with PBS. After that, cells were incubated with NBT/BCIP dye (Cat Nr. 72091-10ML, Sigma-Aldrich) for 20 minutes at room temperature in a dark and washed once with PBS. Images were taken on the Nikon Eclipse TS 100 microscope.
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, Ashland, OR, USA).
Assessment of apoptosis
Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit I (Cat Nr. 556547, Becton-Dickinson) following manufacturer’s instructions. Samples were analyzed using a FACS Canto II flow cytometer (Becton-Dickinson) and FlowJo software (FLOWJO, LLC).
Intracellular NAD+ measurement
Intracellular NAD+ was measured in cell lysates of 1 × 104 cells using the NAD/NADH-Glo™ Assay Kit (Promega). Luminescence was detected with a GloMax®-Multi+ Detection System (Promega).
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. 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 sunless 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).
Statistical analyses were conducted using Student’s t-test or Bootst Ratio19 Statistical significance was taken to be p< 0.05.