It has been proposed that the definitive hematopoietic cell lineages are derived from hemogenic endothelial cells. Recently, CD41 was identified as an earliest cell surface marker of hematopoietic progenitor cells during mouse embryogenesis. We examined relationship between VE-cadherin+ hemogenic endothelial cells and CD41+ progenitors as developmental origins of hematopoietic cells by using in vitro differentiation system of ES cells as well as mouse embryos.

FACS analyses on ES cells differentiating on OP9 stromal cells identified two cell populations, CD41+CD45− and CD41lowVE-cadherin+CD45−. The CD41+ cell population was derived from Flk1+ cells that represent lateral plate mesoderm but not from PDGFRa+ cells that represent paraxial mesoderm. CD41 expression on CD41+CD45− cells was weak at Day4 of ES cell culture. CD41+CD45− cells rapidly increased in number and CD41 expression became higher after Day5. CD45+ cells became detectable as a subpopulation of CD41+ cells two days after the appearance of the CD41+CD45− cell population. A significant proportion of the purified CD41lowVE-cadherin+CD45− cells differentiated to cells with CD41+CD45− phenotype in short-term culture, while CD41+CD45− cells did not differentiate into VE-cadherin+ cells. Unsurprisingly only CD41lowVE-cadherin+CD45− population had potential to produce endothelial cell colonies on OP9 cell layer. Liquid cultures and methylcellulose colony assay with a proper combination of cytokines showed that primitive erythroid colony forming cells were highly enriched in the CD41+CD45− cell population. CD41+CD45− cells also differentiated to Ter119+ definitive erythrocytes and Gr-1+ and Mac-1+ myeloid cells. In contrast, CD41lowVE-cadherin+CD45− cells produced only few hematopoietic cells in the same condition. However, CD41lowVE-cadherin+CD45− cells were capable of differentiating into multi-lineage hematopoietic cells including B lymphocytes when cultured with OP9 stromal cells. CD41+CD45− cells did not show any B lymphogenic potentials even when cultured with OP9 cells.

We examined hemogenic potentials of phenotypically equivalent cells purified from mouse embryos. FACS analyses on cells dissociated from yolk sac and lower trunk of embryos proper revealed two distinct populations, CD41+CD45− cells and CD41−/lowVE-cadherin+CD45− cells. Both populations were already detectable in 8.5 dpc embryos. CD41−/lowVE-cadherin+CD45− cells but not CD41+CD45− cells produced endothelial cell colonies in vitro. The CD41−/lowVE-cadherin+CD45− cell population isolated from yolk sac was able to differentiate into multi-lineage hematopoietic cells when cultured with OP9 cells. However, the same population that was isolated from embryos proper had very poor potential to generate erythroid and myeloid cells although it still initiated robust production of B lymphocytes. Nevertheless, hemogenic activities of this population declined to undetectable level on 11.5 dpc. In contrast, CD41+CD45− cells isolated from yolk sac and embryos proper gave rise to multilineage hematopoietic cells and those potentials were stronger than that of yolk sac-derived CD41−/lowVE-cadherin+CD45− cells. Our results suggest that two distinct precursors, hemogenic endothelial cells and CD41+ progenitor cells, may contribute to the initiation of definitive hematopoiesis in mouse ontogeny, although activity of hemogenic endothelial cells in embryo proper might be unexpectedly limited.

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