B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency

The ontogenic source of definitive hematopoietic system has been identified in non-mammalian vertebrates such as birds and amphibians by orthotopic embryo grafting, but remains unclear for mammals because of technical difficulties. Here, we successfully generated mouse chimeras by grafting yolk sac (YS) on YS of the host embryos before establishing circulation between YS and embryo proper and cultured the whole embryo for 66 h. Donor YS were isolated from C57BL/6 Ly-5.1 and EGFP-transgenic mouse embryos, and recipient embryos from C57BL/6 Ly-5.2 mouse. Almost one-half of the grafts in YS–YS chimeras survived and had obvious blood flow; graft-derived cells comprised 12.7 ± 0.9% of the blood cells in the circulation. These graft-derived blood cells consisted mainly of erythroid cells, some myeloid cells and a few blastic cells. In addition, CD19+ B cells were generated from the graft-derived cells isolated from aorta–gonad–mesonephros (AGM) regions of the YS–YS chimeras; however, the frequency of the YS-derived B cell was low (1.0 ± 0.6%) when co-cultured with OP9 stromal cells. These results demonstrate that B cell potential exists in YS before the circulation. Although the major source for B cell is intra-embryonic AGM region, YS may contribute to definitive lymphopoiesis in vivo in mice.