Legends for Supplemental Figure s
Figure S1. Generation of iPS cells. Human dermal fibroblasts cultured in 6-well plates were infected with Lentivirus vectors to introduce Oct3/4, Sox2, Klf4, and c-Myc. After 6 days, fibroblasts were recovered from the culture plates and transferred onto 90 mm-dishes with primary mouse fibroblast feeder cells. At days 25-28, cell colonies morphologically similar to those of human ES cells were picked up under microscopic observation. About 70 cell clones were picked up and subjected to differentiation culture. More than half of the human iPS cell clones successfully differentiated into floating hematopoietic cells. Among them 2 clones, named WL26 and WL 59, were introduced with an expression vector for Cre-recombinase (pCAGGS-Cre-IRES-Neo) and selected with G418. After 14-15 days, G418-resistant cell colonies were picked up and cultured to expand. PCR analysis revealed deletion of cDNA for Sox-2 and c-Myc in the transgenes (data not shown).
Figure S2. Photo images of iPS cells. iPS cells before or after deletion of transgene-derived Sox-2 and c-Myc cDNA were analyzed for expression of alkaline phosphatase (ALP), TRA-1-81, E-cadherin (E-CAD), SSEA3 and SSEA4.
Figure S3. A schematic depiction of differentiation culture to generate iPS-DC and iPS-MP with OP9 feeder cells and culture media containing fetal calf serum.
Figure S4. A schematic depiction of xeno-free differentiation culture to generate iPS-DC and iPS-MP.
Figure S5. Generation of iPS-DC and iPS-MP by a xeno-free differentiation culture. Phase-contrast images of cells at 1st step d6 (A), 1st step d15 (B), 2nd step d15 (C), and iPS-DC stimulated with TNF-a plus OK432 (D) are shown. (E) May-Grunwald Giemsa staining of iPS-DC on a glass slide is shown. (F) Cell surface expression of CD31, CD45, CD43, and CD11b on cells recovered from 2nd step culture were analyzed. (G) iPS-DC were analyzed for the expression of CD80, CD86, CD83, CD40, HLA class I, and HLA class II. (H) iPS-MP were analyzed for the expression of CD11b, CD14, and CD68.
Figure S6. Function of iPS-DC generated in a xeno-free condition. (A) Indicated numbers of iPS-DC stimulated with OK432 (squares) or with TNF-a plus LPS (diamonds), left unstimulated (triangles), or myeloid cells recovered from 2nd step culture (circles) were X-ray-irradiated and co-cultured with allogeneic peripheral blood T cells (4 x104 cells/well) in a 96-well round-bottomed culture plate for 5 d. Proliferation of T cells was measured based on [3H]-thymidine uptake in the last 16 h of the culture. The data are presented as the mean value +/- SD of duplicate cultures. (B, C) iPS-DC were plated (1 x 105 cells/200 ml/in 96-well culture plates) in the presence or absence of TNF-a(10 ng/ml), LPS (3 mg/ml), or OK432 (10 or 20 mg /ml) as indicated. The supernatant was collected after 60 hours and the concentration of TNF-a (B) and IL-12p70 (C) was measured by ELISA. Data are indicated by the mean value + SD of triplicate cultures.
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