Primary culture systems used in this study.

(A) AGM culture. AGM culture was developed to analyze hematopoiesis of the AGM region in vitro. Tissue of the AGM region isolated from E11.5 embryos was dissociated into a single-cell suspension and cultured in the presence of SCF, bFGF, LIF, and OSM for 10 days. Then, floating hematopoietic cells spontaneously generated in the culture were harvested and analyzed for expression of cell-surface markers and progenitor activities. (B) AGM/FL coculture. The AGM/FL coculture system was designed to analyze the interaction between AGM HSCs and the FL hematopoietic microenvironment. Nonhematopoietic FL cells from E14.5 embryos were first cultured for 2 days to create a hematopoietic microenvironment, and then AGM-derived cells (either whole or CD34⁺/c-Kit⁺ cells) were overlaid. Ten days later, floating hematopoietic cells generated from input cells over the stromal layer were harvested and analyzed as in (A). To distinguish donor cells from recipients, GFP⁺ embryos were used as input cells in both systems.