[Analyzing hematopoietic development of mouse embryonic AGM region by tissue culture]

To analyze hematopoietic kinetics of mouse embryonic aorta-gonad-mesonephros (AGM) region, an in vitro tissue culture method was developed in this study, partly based on previous reports. After 2 days of tissue culture, a significant number of erythro myeloid progenitors, as quantitated by colony forming assay was detected in the AGM region. Moreover, the cells from cultured E10.5 AGM region could generate 10.8 ± 3.5 colony-forming unit in spleen (CFU-S) per tissue on average. Transplantation of cultured E10.5-E11.0 AGM cells resulted in efficient (85.7% repopulated) and long-term (> 4 months) reconstitution of lethally irradiated adult recipients with remarkable chimerism [(51.12 ± 21.17)%]. The multilineage contribution of donor cells was validated by significant engraftment of myeloid and/or lymphoid cells in peripheral blood, bone marrow, spleen and thymus of recipients. Taken together, the tissue culture method can enable us to manipulate the AGM region in vitro, fulfilling a systematic evaluation of developmental kinetics of various hematopoietic precursor cells, particularly HSC, in normal and mutant mid-gestation mouse embryos.