Differentiation of embryonic stem (ES) cells in vitro yield different kinds of hematopoietic progenitors including primitive and definitive hematopoietic cells. It has been reported that HOXB4 induction enable york sac (YS) cells and embryoid body-derived cells to engraft in the irradiated adult mice, however, since the characteristic of ES-derived transplantable cells is not clear, generating hematopoietic stem cells (HSCs) in vitro still remains to be resolved. We previously reported the generation of definitive HSCs from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (Matsuoka, et al; Blood 2001) Co-cultureing on AGM-S3, these YS cells and PS-Sp cells acquired the reconstituting potential of adult irradiated mice. Here we intended to make HSCs using this stromal cells. We differentiated ES cells labeled with GFP on OP9 stromal cell, which is supportive for Hematopoietic differentiation. After 4 days, we sorted Flk1+ cells, which is considered as a marker of hemangilblasts, and transferred them onto A-9, subline of AGM-S3, or OP9 stromal cell layer with cytokines. After several days incubation, we examined the emergence of CD34+ c-kit+ cells and colony forming ability of CD34+ or CD34 cells. CD34+ cells contained more CFU-Mix than CD34 cells. When compared on A-9 or OP9, cultured cells on OP9 contained more CFU activity than on A-9. We sorted and cultured CD34+ c-kit+ cells on OP9 for 7–10 days, and confirmed Ter119+, Gr-1+, or Mac-1+ cells differentiated from CD34+ c-kit+ cells by FACS analysis. Next, we cultured Flk1+ cells on A-9 or OP9 for 7–15 days and transplanted all the collected cells into 2.4Gy irradiated NOD-SCID mice. After 3 months after transplantation, FACS analysis showed no GFP+ cells in the recipient BM. However, PCR analysis detected donor derived DNAs in BM when Flk1+ cells were cocultured on A-9. We next transplanted 1×104 of CD34+ CD45+ or CD34+ CD45 cells from Flk1+ cells cocultured on A-9 or OP9 into 2.4 Gy irradiated NOD-SCID mice. PCR analysis revealed donor derived DNAs in mice transplanted with CD34+ CD45+ cells on A-9 and with CD34+ CD45 cell on OP9. These data suggested that CD34+ cells differentiated from Flk1+ cells have powerful hematipoietic activity and showed different potential cultured between on A-9 and on OP9.

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