Undifferentiated human ESC and iPSC lines were cultured on mitotically-inactivated SNL feeder cells with Primate ES cell medium supplemented with 4 ng/ml bFGF. During the differentiation of the cells into macrophages, cells were cultured under 37°C , with 5% CO2 and 5% O2. On day 0, the iPSCs were plated at a ratio of 1:15 onto a mitotically-inactivated OP9 feeder layer on 100 mm cell culture plates in αMEM (Invitrogen) containing 10% FBS and 1% Antibiotic-Antimycotic (Invitrogen) supplemented with 50 ng/ml VEGFα (R&D Systems). On day 5, the medium was changed. On day 10, the differentiating iPSCs were collected by trypsinization, and Tra-1-85+ CD34+ and KDR+ hematopoietic progenitors were sorted on a FACS Aria II instrument (BD Biosciences). The progenitors were plated at 2 x 104 cells on another mitotically inactivated OP9 feeder layer on 100 mm cell culture plates or at 3 x 103 cells/well in 6 well cell culture plates in αMEM containing 10% FBS and 1% Antibiotic-Antimycotic supplemented with 50 ng/ml IL-3, 50 ng/ml SCF, 10 ng/ml TPO, 50 ng/ml FL and 50 ng/ml M-CSF (all R&D Systems). On day 18, the medium was changed. On day 26, differentiating cells were collected with Accumax (Innovative Cell Technologies), and CD14+ iPS-MPs were purified on an autoMACSpro instrument (Miltenyi Biotec).
We expanded the fibroblasts in DMEM (Nacalai tesque) containing 10% FBS (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen). To generate iPS cells, we introduced OCT3/4, SOX2, KLF4 and c-MYC by using ecotropic retroviral transduction into fibroblasts expressing the mouse Slc7a1 gene. Six days after transduction, the cells were harvested and re-plated onto mitotically inactivated SNL feeder cells. The next day, we replaced the medium with Primate ES cell medium (ReproCELL) supplemented with 4 ng/ml bFGF (Wako). Three weeks after this period, individual colonies were isolated and expanded. Cell culture was performed under 37°C , with 5% CO2 and 21% O2.
Total RNA was column-purified with the RNeasy kit (Qiagen) and treated with RNase-free DNase (Qiagen).
20 µg of total RNA was first amplified with WT-OvationTM Pico System (Nugen). The RNA was labelled with Cy3 using Genomic DNA Enzymatic Labeling Kit (Agilent Technologies).
Labelled cDNA was fragmented with Gene Expression Hybridization Kit (Agilent Technologies), followed by hybridization at 65˚C for 17 hrs.
Images were acquired by DNA Microarray Scanner (Agilent Technologies)
ES cells which was previously established
Raw data were processed with FeatureExtraction version 10.7. Background signal was subtracted.