Expansion of fetal liver hematopoietic stem cells on AGM-derived endothelial cell lines. (A) Three thousand fetal liver stem cells (lin⁻CD34⁺Sca-1⁺c-Kit⁺) were plated on each stromal cell line and cultured for 7 days. Cells were trypsinized, and nonadherent cells were collected and counted. (B) Cell contact is required for efficient expansion of fetal liver stem cells on DAS 104-4. One thousand fetal liver stem cells (lin⁻⁻CD34⁺Sca-1⁺c-Kit⁺) were plated either directly onto the DAS 104-4 cell line or in a transwell (insert) immersed in media conditioned by this cell line. After 1 week, nonadherent hematopoietic cells were isolated and counted. (C) Hematopoietic colony morphologies of fetal liver stem cells plated on the DAS 104-4 cell lines after 5 days in culture. Panels a (20×) and c (200×) show a “cobblestone”-type structure containing blast-like cells (arrow in panel a). Panels b (20×)and d (200×) show macrophage-like cells that predominate in the cultures.