Development of functional macrophages from embryonal stem cells in vitro.

When cultured under appropriate in vitro conditions, embryonal stem cells (ESCs) form embryoid bodies (EBs) that contain mature hematopoietic cells, including cells of the monocyte-macrophage lineage. A two-step in vitro culture system for generation of ESC-derived macrophages has been developed and optimized. Maximum numbers of macrophage-containing colonies developed in secondary hematopoietic cultures of cells from disrupted EBs after 9 to 12 days of differentiation when interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) were included in both primary and secondary cultures. Over 10(5) viable, phagocytically active macrophages were generated from cultures initiated by 7500 ESCs. The inclusion of stem cell factor (SCF) in primary cultures not only increased the frequency of progenitor cells but also the cellular heterogeneity of colonies. SCF in secondary cultures increased the cellularity, but not the frequency, of macrophage-containing colonies; although cellular heterogeneity was also increased, there was still an overall increase in yield of macrophages.