Cd14 monocytes

Two types of human monocyte-derived macrophages (h-Mo-Mφs) were generated as described in Supplementary Figure S1A. CD14⁺ monocytes (AllCells, Alameda, CA, USA) were cultured (37°C, 5% CO₂) in RPMI1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, Bergisch Gladbach, Germany) or 100 ng/ml human macrophage colony-stimulating factor (M-CSF; R&D Systems) for 7 days. The M-CSF-treated cells were further cultured in RPMI1640 supplemented with 10% FBS, 100 ng/ml human M-CSF, and 20 ng/ml human interleukin (IL)-4 (R&D Systems) for 2 days. Human monocyte-derived DCs (h-Mo-DCs) were obtained as shown in Supplementary Figure S2B. For h-Mo-DC generation, CD14⁺ monocytes were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 100 ng/ml human GM-CSF and 100 ng/ml human IL-4. Cells were incubated at 37°C in an atmosphere containing 5% CO₂ for 6 days, with medium changes on days 2 and 3. Generated cells were harvested on day 6 and reseeded in fresh medium supplemented with 100 ng/ml GM-CSF and 100 ng/ml IL-4. To generate mature DCs, cells were further supplemented with 10 μg/ml CD40 agonist antibody (patent, WO/2002/088186). The characteristics of monocyte-derived cells were verified by flow cytometry (FCM).

All iPSC lines were cultured with mTeSR1 (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) on Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) -coated 6-well plates. Cell lines OP9, T98G, U-87, MCF7, BT-474, MDA-MB-453, Daudi, Hep G2, SK-OV-3, SCC-25, Raji, FHs 74 Int, HCT 116, Malme-3, Malme-3M, RPMI 8226, K562, THP-1, SW480, MCF 10A, MRC-5, NCI-H460 [American Type Culture Collection (ATTC), Manassas, VA, http://www.atcc.org] were cultured as recommended by ATCC. Cell line FM-57 [European Collection of Authenticated Cell Cultures (ECACC)] was culture cultured in RPMI 1640 with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com). Cell line OP9-DLL1 (Riken BRC Cell Bank, Ibaraki, Japan, http://cell.brc.riken.jp/en/) was cultured in MEMα (Thermo Fisher Scientific) with 20% FBS. Frozen human peripheral blood CD14+ monocytes (Lonza, http://www.lonza.com) were thawed and maintained in RPMI 1640 with 10% FBS.