Development of hESC-derived hematopoietic progenitor cells with expression of functional Notch-1. (A) Time course of hematopoietic progenitor cell production by hESCs. Coculture of undifferentiated hESCs with S17 stromal cells for the indicated number of days was followed by flow cytometric analysis of surface expression of CD34 and CD45. These results demonstrate surface antigen expression both on the unsorted cell population and the enriched CD34⁺ cell population. (B) hESC-derived cells were harvested after 14 days of culture on S17 stromal cells and analyzed by flow cytometry for expression of CD34 and Notch-1. The data were electronically gated to distinguish CD34⁺ cells (solid line) from CD34⁻ nonhematopoietic cells (dashed line). Isotype control staining is also indicated (filled histogram). (C) After 20 days of culture on S17 cells, total RNA was prepared from unsorted hESC/S17 cells or from the indicated populations of CD34⁺, CD34⁻, CD45⁺, or CD45⁻ cells. cDNA was prepared by reverse transcription, and expression of Notch-1, HES-1, and Actin was analyzed by PCR.