Decreased expression of Notch1 gene in CD34⁺ cells cocultured with HS-27a and monocytes. (A) CD34⁺ cells were isolated from apheresis products obtained from G-CSF-mobilized healthy donors. The CD34⁺ cells were seeded in a Transwell insert and cultured in media containing G-CSF, IL-3, IL-6, SCF, and 10% FCS. The bottom chamber contained either no cells (control), HS-27a cells (+HS-27a), CD14⁺ cells (+CD14), or HS-27a plus CD14⁺ cells (+HS-27a and CD14). The CD34⁺ cells were harvested after 4 days, and total RNA was isolated for determination of Notch1 gene expression by a Syber Green real-time PCR assay, normalized to G3PDH gene expression. (B) Conditioned media from the above cultures were collected, and OPN secretion was measured by ELISA. (C-D) The CD34⁺ cells were seeded in a Transwell insert and cultured in the presence or absence of 1 μg/mL OPN in addition to the growth factor cocktail for 4 days (+OPN or control, respectively). (C) Notch1 and (D) integrin alpha 6 (ITGA6) gene expression were determined by a Syber Green assay as described for panel A. The data represent the average ± SD for at least 2 samples.