FIG. 6.

Primary cell characterization. (A) Undifferentiated hESCs and hEBs. Undifferentiated hESC culture, phase contrast, bar = 100 μm; Oct 4-stained undifferentiated hESC colony, bar = 50 μm; Nanog-stained undifferentiated hESC colony, bar = 50 μm; hEBs, day 10, bar = 500 μm. (B) Flow cytometry analysis of undifferentiated hESCs. SSEA-1, SSEA-3, SSEA-4, and Tra-1-81 (inset is the isotype control) were used to confirm the features of ESCs. (C) Flow cytometry analysis of hEBs in EB medium plus cytokines plus BMP-2 at day 10 and day 15. Hematopoietic markers: CD31 (y axis), CD34, and CD45. Insets are isotype controls. (D) Hematopoietic stem cells (HSC) do not produce TNF-α by ELISA. Cells were stimulated with PMA and ionomycin, 1 μg/ml of LPS, or diluent (NS) for 6 h. TNF-α was measured by ELISA. (E) Cells from early differentiation states do not produce TNF-α message. Cells were mock treated or treated with 100 ng/ml PMA and 500 ng/ml of ionomycin for 3 h. RNA was isolated and reverse transcribed. The untreated ESCs were used as the calibrator. cDNA quantity is expressed as increase (fold) over unstimulated ESCs. (F) Peripheral blood monocytes are largely competent for the production of TNF-α. Two different preparations of monocytes stimulated with LPS are shown using intracellular flow cytometry.